Methods for conducting metabolic analyses

ABSTRACT

The present invention provides methods and apparatus for purifying metabolites of interest and conducting metabolic analyses. The methods generally involve determining metabolic flux values for a plurality of target analytes by monitoring the relative isotope abundance of a stable isotope in a substrate labeled with the stable isotope and/or one or more target metabolites formed through metabolism of the labeled substrate. Certain methods utilize multiple electrophoretic methods to separate the target analytes from other components within the sample being analyzed. The methods can be used in a variety of applications including screens to identify metabolites that are correlated with certain diseases and diagnostic screens for identifying individuals having, or susceptible to, a disease.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/687,909, filed Oct. 17. 2003, now U.S. Pat. No. 6,849,396 which is acontinuation of U.S. application Ser. No. 09/553,424, filed Apr. 19,2000, now U.S. Pat. No. 6,764,817 which claims the benefit of U.S.provisional application 60/130,238, filed Apr. 20, 1999. Thisapplication is also related to U.S. provisional application 60/075,715filed Feb. 24, 1998; copending U.S. patent application number Ser. No.09/513,486, filed Feb. 25, 2000; copending U.S. patent application Ser.No. 09/513,395, filed Feb. 25, 2000; copending U.S. application Ser. No.09/513,907, filed Feb., 25,2000; copending U.S. patent application Ser.No. 09/551,937, filed Apr. 19, 2000; and copending PCT application PCT/US01/10504, filed Apr. 19, 2000. All of these applications areincorporated by reference in their entirety for all purposes.

FIELD OF THE INVENTION

This invention relates to the field of metabolism and separationtechnology, including methods for separating and analyzing metabolitesand making correlations between certain metabolites or metabolicconditions and cellular states.

BACKGROUND OF THE INVENTION

One goal in biochemical research is to develop correlations between thepresence, absence, concentration, conversion rates, or transport ratesof certain molecules within cells, tissues and particular cell or tissuestates (e.g., disease states, particular developmental stages, statesresulting from exposure to certain environmental stimuli and statesassociated with therapeutic treatments). Such correlations have thepotential to provide significant insight into the mechanism of disease,cellular development and differentiation, as well as in theidentification of new therapeutics, drug targets and/or disease markers.

Genomics based studies are an example of one type of approach taken insuch investigations. Typically, functional genomics focuses on thechange in mRNA levels as being indicative of a cellular response to aparticular condition or state. Recent research, however, hasdemonstrated that often there is a poor correlation between geneexpression as measured by mRNA levels and active gene product formed(i.e., protein encoded by the mRNA). This finding is not particularlysurprising since many factors—including differences in translationalefficiency, turnover rates, extracellular expression orcompartmentalization, and post-translational modification affect proteinlevels independently of transcriptional controls.

Another approach is proteomics which, as the term implies, focuses onthe proteins present in various cellular states. The rationale forconducting proteomics investigations is based in part upon the view thatcertain aspects of cellular biology can be better understood by takinginventory of protein levels rather than nucleic acids levels,particularly given the findings just described that suggest that proteinactivity often hinges on factors other than the concentration of mRNAencoding the protein.

Instead of focusing exclusively on either nucleic acids or proteins, thecurrent invention takes a different approach and examines themetabolites present in a cell formed through cellular metabolism. Suchan approach is termed metomics. More specifically, metomics refers tothe study of metabolic fluxes and changes in these fluxes as a functionof the physiological state of an organism (or population of cells ortissue). Metomics studies can involve, for example, identifying specificmetabolic patterns that cause or result from changes in thephysiological state of an organism or cell population. Metomics studiescan be correlated to changes in protein and mRNA expression patternsalso resulting from changes in the physiological state of an organism orcell population.

Metabolism consists of a complex network of catabolic (energy andprecursor producing) and anabolic (biosynthetic) enzymatic pathways thattogether support the maintenance and growth of the cell. The flow ofchemicals through this network of enzymatic reactions ovaries with thecell cycle; (Ingraham, J. L., et al., Growth of the Bacterial Cell,Sinauer Associates, Sunderland, Mass., (1983)) diet, availability ofextracellular nutrients, and exposure to cellular stresses (e.g.,chemical and biochemical toxins or infectious agents). The majormetabolic pathways and factors in their regulation are discussed in anygeneral biochemical text book including, for example, Voet, D. and Voet,J. G., Biochemistry, John Wiley & Sons, New York (1990); Stryer, L.,Biochemistry, 2nd ed., W.H. Freeman and Company, San Francisco (1981);and White, A., et al., Principles of Biochemistry, 6th ed., McGraw-HillBook Company (1978), each of which is incorporated by reference in itsentirety.

Because metabolism must be capable of adapting to varying conditions andstimuli, cells have a variety of mechanisms at their disposal toregulate metabolism. For example, certain regulatory mechanisms controlthe rate at which metabolites enter a cell. Since very few substancesare capable of diffusing across a cellular membrane, such regulationtypically occurs via one of the active or passive transport mechanismsof a cell.

In addition to transport control, a number of different mechanisms canfunction to regulate the activity of an enzyme that is part of ametabolic pathway. For example, a product produced by the enzyme can actvia feedback inhibition to regulate the activity of the enzyme. Enzymescan also be regulated by ligands that bind at allosteric sites (i.e.,sites other than the active site of the enzyme). It has been suggestedthat allosteric regulation is important in quick time responses (timesless than that required for the induction and synthesis of new proteins,<10 min), as well as in the modulation of enzyme activity to changes inbackground requirements (feed-back control) (Chock, P. B., et al.,Current Topics in Cellular Regulation., 27:3 (1985); Koshland, D. E., etal., Science, 217:220 (1982); Stadtman, E. R. and Chock, P. B., CurrentTopics in Cellular Regulation, 13:53 (1978)). Allosteric regulation isthe primary method used by bacteria to sense their environment, both byactivity modulation of already synthesized proteins and by eliciting newprotein synthesis via control of RNA polymerase promoter and repressorproteins (Monod, J., et al., J. Mol. Biol., 6:306 (1963)). Allostericregulation can be associated with multimeric proteins (several subunitsworking in a concerted fashion) and/or within regulatory cascades inorder to: (1) provide more sites for different regulatory ligands toaffect activity, (2) amplify the rate of response, (3) amplify themagnitude of response, and/or (4) amplify the sensitivity of response(Chock, P. B., et al., Current Topics in Cellular Regulation., 27:3(1985); Koshland, D. E., et al., Science, 217:220 (1982); Stadtman, E.R. and Chock, P. B., Current Topics in Cellular Regulation, 13:53(1978)).

Expression regulation constitutes another metabolic regulatorymechanism. Concerted sets of genes, encoding small numbers of proteins,are often organized under the same transcriptional control sequencecalled an operon. However, where the necessary adaptive changes entailthe induction of large numbers of proteins, many such operons can belinked in regulons. For example, in E. coli the following stimuli inducethe number of proteins indicated in parentheses: (a) heat shock (17proteins), (b) nitrogen starvation (≧5 proteins), (c) phosphatestarvation (≧82 proteins), (d) osmotic stress (≧12 proteins), and (e)SOS response (17 proteins) (see, Neidhardt, F. C., in: Escherichia coliand Salmonella typhimurium: cellular and molecular biology, F. C.Neidhardt et al. (eds.), pg. 3, Amer Soc Microbiology, Washington, D.C.,(1987); Neidhardt, F. C. and Van Bogelen, R. A., in: Escherichia coliand Salmonella typhimurium Cellular and Molecular Biology., F. C.Neidhardt (ed.)., pg 1334, American Society of Microbiology, Washington,D. C., (1987); Magasanik, B. and Neidhardt, F. C., in Escherichia coliand Salmonella typhimurium Cellular and Molecular Biology., F. C.Neidhardt (ed.), pg 1318, American Society of Microbiology, Washington,D.C., (1987); (VanBogelen, R. A., et al., Electrophoresis, 11:1131(1990)); Wanner, B. L., in: Escherichia coli and Salmonella typhimuriumCellular and Molecular Biology, F. C. Neidhardt (ed.), pg 1326, AmericanSociety of Microbiology, Washington, D.C., (1987)); (Christman, M. F.et. al, Cell, 14:753 (1985); and Walker, G. C., in Escherichia coli andSalmonella typhimurium Cellular and Molecular Biology, F. C. Neidhardt(ed.), pg 1346, American Society of Microbiology, Washington, D.C.,(1987)). Thus, regulons enable cells to regulate genes that need torespond occasionally in a concerted fashion to a particular stimulus,but that at other times need to be independently responsive toindividual controls (Neidhardt, F. C., in: Escherichia coli andSalmonella typhimurium: cellular and molecular biology, F. C. Neidhardtet al. (eds.), pg. 3, Amer Soc Microbiology, Washington, D.C., (1987)).

Degradation is another regulatory mechanism for controlling metabolism.Most proteins are very stable, at least under conditions of balancedgrowth, probably because the cell pays such a high price to make them.However, several researchers have observed a limited class of cellularproteins (10 to 30% of the total protein present during exponentialgrowth in bacteria) that is unstable (exhibit half-lives of 60 min orless). Proteins within the class appear to be turned over quickly within10 hours of any growth down shift, and during exponential growth (NathK. and Koch. A. L., J. Biol. Chem., 246:6956 (1971); St. John, A. C. andGoldberg, A. L., J. Bacteriol., 143:1223 (1980)). At least some of theselabile proteins, during energy and nutrient down-shifts, are proteins ofthe protein synthesizing system (e.g., ribosomal proteins) (Davis, B.D., et al., J. Bacteriol., 166:439 (1986)); Ingraham, J. L., et al.,Growth of the Bacterial Cell, Sinauer Associates, Sunderland, Mass.,(1983); Maruyama, H. B. and Okamura, S., J. Bacteriol., 110:442 (1972)).This conclusion is drawn from the observations that the apparent rate ofprotein synthesis per unit of protein synthesizing proteins decreases atlow growth rates, but the time required for the initial synthesis ofinducible enzymes remains constant at all growth rates (Ingraham, J. L.,et al., Growth of the Bacterial Cell, Sinauer Associates, Sunderland,Mass. (1983)).

Given the interrelatedness between different cell states and metabolismand the fact that the focus of metomics differs from genomics andproteomics, the present invention utilizes metomic studies to gain newinsight into the correlation between cellular states and thebiomolecules within the cell.

SUMMARY OF THE INVENTION

The present invention provides apparatus and methods that have utilityin purifying and detecting metabolites of interest. The purifying anddetection methods enable one to determine how various parameters formetabolites of interest (e.g., metabolite concentration and/or flux)vary as a function of different cellular states or exposure to differentstimuli. Thus, the methods can be used to screen for metabolites thatare correlated with particular cellular states or stimuli. Suchinformation can be used to develop metabolic “fingerprints” or“profiles” that are characteristic of different cellular states and/orresponses to particular stimuli. The information can also be used todevelop metomics databases. Once correlations, have been established,certain methods of the invention can be utilized to screen forparticular states. For example, some methods screen individuals toidentify those having, or at risk, for a particular disease based uponsimilarities between their metabolic profile and that of diseased and/orhealthy individuals.

More specifically, the invention includes various separation methods.Certain methods involve performing a plurality of capillaryelectrophoresis methods in series. Each method in the series includeselectrophoresing a sample containing multiple metabolites andpotentially one or more target analytes of interest so that a pluralityof resolved metabolites are obtained. The sample electrophoresed in eachmethod contains only a subset of the plurality of resolved metabolitesfrom the immediately preceding method in the series, except the firstmethod of the series in which the sample is the initial sample.Fractions containing resolved metabolites from the final electrophoreticmethod are analyzed to detect the presence of the target analytes. Thecapillary electrophoresis methods within the series are selected fromthe group consisting of capillary isoelectric focusing electrophoresis,capillary zone electrophoresis and capillary gel electrophoresis.

In certain aspects, the invention provides various methods for analyzingmetabolic pathways. Certain methods involve administering a substratelabeled with a stable isotope to a subject, the relative isotopicabundance of the isotope in the substrate being known prior toadministering the substrate. The subject is then allowed sufficient timeto at least partially metabolize the labeled substrate to form one ormore target metabolites. The abundance of the isotope in a plurality oftarget analytes in a sample taken from the subject is then determined sothat a value for the flux of each target analytes can be ascertained.The multiple target analytes for which a flux value is determined areeither the substrate and/or one or more target metabolites. Theabundance of the isotope in the target analytes is determined using ananalyzer capable of determining the ratio of the isotopically enrichedisotope to the more abundant isotope (e.g., ¹²C/¹³C, ¹⁴N/¹⁵N, ¹⁶O/¹⁸Oand ³⁴S/³²S). Examples of such analyzers include mass spectrometers,infrared spectrometers and nuclear magnetic resonance spectrometers.

Prior to determining the abundance of the isotope in the target analytesand corresponding flux values, typically the target analytes are atleast partially separated from other components in the sample. Generallythis is accomplished by performing a plurality of electrophoreticseparation methods in series, such that samples from fractions obtainedafter one method are used in a subsequent electrophoretic method. Theactual electrophoretic methods employed can vary, but typically includecapillary isoelectric focusing electrophoresis, capillary zoneelectrophoresis and capillary gel electrophoresis. In some instances,separation and elution conditions of the electrophoretic methods arecontrolled so that separate fractions for one or more classes ofmetabolites (e.g., proteins, polysaccharides, carbohydrates, nucleicacids amino acids, nucleotides, nucleosides, fats, fatty acids, andorganic acids) are obtained. This simplifies the analysis because onecan simply analyze those fractions containing the class of components towhich the target analytes belong.

The invention also provides analytic methods for analyzing metabolicpathways in which samples from a subject have been previously obtained.In such instances, certain methods involve separating at least partiallya plurality of target analytes from other components contained in thesample obtained from the subject. The target analytes comprise asubstrate labeled with a stable isotope and/or one or more targetmetabolites resulting from the metabolism of the substrate by thesubject. A flux value for each target analyte is determined fromknowledge of the isotopic abundance in the substrate prior to it beingadministered to the subject and by determining the abundance of theisotope in the target analytes.

Methods for screening metabolites to identify those correlated withvarious cellular states (e.g., certain diseases) are also included inthe invention. Certain screening methods include administering asubstrate labeled with a stable isotope to a test subject and a controlsubject, the relative isotopic abundance of the isotope in the substratebeing known and the test subject having a disease under investigation.The labeled substrate is allowed to be at least partially metabolized bythe test subject and control subject to form one or more targetmetabolites. The conditions under which the administering and allowingsteps are performed are controlled so that they are the same for thetest and control subject. A sample is obtained from the test and controlsubject and the relative abundance of the isotope in the target analytesdetermined to obtain a value for the flux of each target analyte. Theflux values for the test and control subject are compared, a differencein the flux value for a target analyte in the test subject andcorresponding flux value for the control subject indicating that suchanalyte is potentially correlated with the disease being studied.

When a sample has been previously acquired, certain screening methodsinvolve analyzing a sample from a test subject having a disease, thesample comprising a substrate labeled with a stable isotope administeredto the test subject and/or one or more target metabolites resulting frommetabolism of the substrate by the test subject The relative isotopicabundance of the isotope in the substrate is known at the time ofadministration, and the analyzing step includes determining the isotopicabundance of the isotope in a plurality of target analytes in the sampleto determine a value for the flux of each target analyte. Flux valuesfor the target analytes in the test subject are compared with fluxvalues for a control subject, a difference in a flux value indicatingthat such analyte is correlated with the disease.

In another aspect, the invention includes methods for screening for thepresence of a disease. Certain of these methods involve administering toa test subject a substrate labeled with a stable isotope, the relativeabundance of the isotope in the substrate being known. Sufficient timeis allowed for the labeled substrate to be at least partiallymetabolized by the test subject to form one or more target metabolitesknown to be correlated with the disease. A plurality of electrophoreticmethods are performed in series to at least partially separate aplurality of target analytes from other biological components in asample obtained from the test subject, the target analytes comprisingthe substrate and/or one or more of the target metabolites. Flux valuesfor the target analytes are determined from the abundance of the isotopein that analyte.

The method is simplified when sample is-provided. In such instances,certain method include analyzing a sample from a test subject, thesample comprising a substrate labeled with a stable isotope administeredto the test subject and/or one or more target metabolites resulting frommetabolism of the substrate by the test subject, the relative isotopicabundance of the isotope in the substrate known at the time ofadministration. The analyzing step itself comprises determining theabundance of the isotope in a plurality of analytes in the sample todetermine a value for the flux of each analyte, the plurality ofanalytes comprising the substrate and/or one or more of the targetmetabolites. For each target analyte, the determined flux value iscompared with a corresponding reference flux value for the same targetanalytes to assess the test subject's risk of disease. The referencevalue can be representative of a healthy or diseased state.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of one example of anelectrophoretic system that can be utilized with certain methods of theinvention.

FIG. 2A is a schematic representation of some of the major elements ofan electrophoretic system utilized in conducting certain electrophoreticmethods of the invention.

FIG. 2B is a cross-sectional view of a capillary showing the orientationof a porous plug inserted into the capillary to control electroosmoticflow in certain methods of the invention.

FIGS. 3A and 3B are top-views of certain elements of microfluidicdevices that can be utilized to conduct certain electrophoretic methodsof the invention.

FIG. 4 is an electropherogram for a sample containing five unlabeledproteins (hen egg white conalbumin, bovine serum albumin, bovinecarbonic anhydrase II, carbonic anhydrase II, rabbit muscle GAPDH, andbovine ribonuclease A) as obtained following electrophoresis bycapillary zone electrophoresis. Absorbance was monitored at 214 nm.Under the conditions of this particular experiment (see Example 1) inwhich the proteins were unlabeled, the proteins were not resolved.

FIG. 5 is a plot of electrophoretic mobility for each of the fiveproteins listed in FIG. 4 under the same electrophoresis conditions asdescribed in FIG. 4.

FIG. 6 is a plot showing the correlations between electrophoreticmobility and the predicted mass-to-charge ratio of the proteins at pH4.0.

FIG. 7 is an electropherogram obtained during separation of a samplecontaining five sulfophenylisothiocyanate-labeled proteins (hen eggwhite conalbumin, bovine serum albumin, bovine carbonic anhydrase II,carbonic anhydrase II, rabbit muscle GAPDH, and bovine ribonuclease A)as obtained following electrophoresis by capillary zone electrophoresis.Absorbance was monitored at 214 nm. Under the conditions of thisparticular experiment (see Example 2) in which the proteins werelabeled, the labeled proteins were partially resolved.

FIG. 8 is an electropherogram obtained during separation of a samplecontaining the proteins hen white conalbumin, bovine serum albumin, andbovine carbonic anhydrase II, by CIEF.

FIG. 9 is an electropherogram of a fraction (fraction F) obtained fromthe separation by CIEF shown in FIG. 7.

FIG. 10 is an electropherogram of a fraction (fraction G) obtained fromthe separation by CIEF shown in FIG. 7.

FIG. 11 shows (A) a section of the mass spectrum around 136.88 amu; (B)a similar section of the mass spectrum exactly 6.02 amu higher,corresponding to the position where the [¹³C]₆-metabolite peak shouldexist; (C) the ratio of the counts at 142.90 to that at 136.88 amupositions in the mass spectrum, corresponding to the six carbon ¹³C/¹²Cmetabolite ratio; and (D) the metabolic flux, determined by curve fit tothe change in the ¹³C/¹²C ratio over time at 136.88 amu.

FIG. 12 shows (A) a section of the mass spectrum around 150.87 amu; (B)a similar section of the mass spectrum exactly 6.02 amu higher,corresponding to the position where the [¹³C]₆-metabolite peak shouldexist; (C) the ratio of the counts at 156.89 to that at 150.88 amupositions in the mass spectrum, corresponding to the six carbon ¹³C/¹²Cmetabolite ratio; and (D) the metabolic flux, determined by curve fit tothe change in the ¹³C/¹²C ratio over time at 150.87 amu.

FIG. 13 shows (A) a section of the mass spectrum around 152.88 amu; (B)a similar section of the mass spectrum exactly 6.02 amu higher,corresponding to the position where the [¹³C]₆-metabolite peak shouldexist; (C) the ratio of the counts at 152.90 to that at 152.88 amupositions in the mass spectrum, corresponding to the six carbon ¹³C/¹²Cmetabolite ratio; and (D) the metabolic flux, determined by curve fit tothe change in the ¹³C/¹²C ratio over time at 152.88 amu.

FIG. 14 shows (A) a section of the mass spectrum around 281.77 amu; (B)a similar section of the mass spectrum exactly 6.02 amu higher,corresponding to the position where the [¹³C]₆-metabolite peak shouldexist; (C) the ratio of the counts at 287.79 to that at 281.77 amupositions in the mass spectrum, corresponding to the six carbon ¹³C/¹²Cmetabolite ratio and (D) the metabolic flux, determined by curve fit tothe change in the ¹³C/¹²C ratio over time at 281.77 amu.

FIG. 15 shows (A) a section of the mass spectrum around 328.76 amu; (B)a similar section of the mass spectrum exactly 6.02 amu higher,corresponding to the position where the [¹³C]₆-metabolite peak shouldexist; (C) the ratio of the counts at 334.78 to that at 328.76 amupositions in the mass spectrum, corresponding to the six carbon ¹³C/¹²Cmetabolite ratio; and (D) the metabolic flux, determined by curve fit tothe change in the ¹³C/¹²C ratio over time at 328.76 amu.

FIG. 16 shows (A) a section of the mass spectrum around 330.76 amu; (B)a similar section of the mass spectrum exactly 6.02 amu higher,corresponding to the position where the [¹³C]₆-metabolite peak shouldexist; (C) the ratio of the counts at 336.78 to that at 330.76 amupositions in the mass spectrum, corresponding to the six carbon ¹³C/¹²Cmetabolite ratio; and (D) the metabolic flux, determined by curve fit tothe change in the ¹³C/¹²C ratio over time at 330.76 amu.

FIG. 17 shows (A) a section of the mass spectrum around 494.84 amu; (B)a similar section of the mass spectrum exactly 6.02 amu higher,corresponding to the position where the [¹³C]₆-metabolite peak shouldexist; (C) the ratio of the counts at 500.86 to that at 494.84 amupositions in the mass spectrum, corresponding to the six carbon ¹³C/¹²Cmetabolite ratio; and (D) the metabolic flux, determined by curve fit tothe change in the ¹³C/¹²C ratio overtime at 494.82 amu.

FIG. 18 shows (A) a section of the mass spectrum around 278.80 amu; (B)a similar section of the mass spectrum exactly 5.02 amu higher,corresponding to the position where the [¹³C]₅-metabolite peak shouldexist; (C) the ratio of the counts at 283.82 to that at 278.80 amupositions in the mass spectrum, corresponding to the five carbon ¹³C/¹²Cmetabolite ratio; and (D) the metabolic flux, determined by curve fit tothe change in the ¹³C/¹²C ratio over time at 278.80 amu.

FIG. 19 shows (A) a section of the mass spectrum around 280.80 amu; (B)a similar section of the mass spectrum exactly 5.02 amu higher,corresponding to the position where the [¹³C]₅-metabolite peak shouldexist; (C) the ratio of the counts at 285.82 to that at 280.8 amupositions in the mass spectrum, corresponding to the five carbon ¹³C/¹²Cmetabolite ratio; and (D) the metabolic flux, determined by cure fit tothe change in the ¹³C/¹²C ratio over time at 280.80 amu.

DETAILED DESCRIPTION

I. Definitions

Unless defined otherwise, all technical and scientific terms used hereingenerally have the same meaning as commonly understood by one ofordinary skill in the art to which this invention belongs. Standardtechniques are used for nucleic acid and peptide synthesis. Generally,enzymatic reactions and purification steps are performed according tothe manufacturer's specifications. The techniques and procedures aregenerally performed according to conventional methods in the art andvarious general references (see generally, Sambrook et al. MOLECULARCLONING: A LABORATORY MANUAL, 2d ed. (1989) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., which is incorporated hereinby reference in its entirety), that are provided throughout thisdocument.

Discussions of the various classes of metabolic compounds referencedherein can be found in any general biochemistry text book, including,for example, Voet, D. and Voet, J. G., Biochemistry, John Wiley & Sons,New York (1990); Stryer, L., Biochemistry, 2nd ed., W.H. Freeman andCompany, San Francisco (1981); and White, A., et al., Principles ofBiochemistry, 6th ed., McGraw-Hill Book Company (1978), each of which isincorporated by reference in its entirety.

A “nucleic acid” is a deoxyribonucleotide or ribonucleotide polymer ineither single- or double-stranded form.

A “polynucleotide” refers to a single- or double-stranded polymer ofdeoxyribonucleotide or ribonucleotide bases.

As used herein, the terms “protein”, “peptide” and “polypeptide” areused interchangeably and refer to a polymer of amino acid residues. Fora general review, see, Spatola, A. F.. in CHEMISTRY AND BIOCHEMISTRY OFAMINO ACIDS, PEPTIDES AND PROTEINS, B. Weinstein, eds., Marcel Dekker,New York, p. 267 (1983), which is incorporated by reference in itsentirety. As used herein, the twenty conventional amino acids and theirabbreviations follow conventional usage (Immunology—A Synthesis. 2nded., (E. S. Golub and D. R. Gren, Eds.) Sinauer Associates, Sunderland,Mass. (1991)). In the polypeptide notation used herein, the lefthanddirection is the amino terminal direction and the righthand direction isthe carboxy-terminal direction, in accordance with standard usage andconvention.

A “carbohydrate” refers to aldehyde or ketone derivatives of polyhydricalcohols. The term includes monosaccharides, oligosaccharides andpolysaccharides. “Oligosaccharides” and “polysaccharides” are formed bycondensation of monosaccharide residues. Oligosaccharides contain arelatively limited number of monosaccharide residues, and typicallyinclude di-, tri-, tetra- and pentasaccharides. Polysaccharides arepolymers of high molecular weight formed from the condensation of manymonosaccharides of the same type (homopolysaccharides) or two or moretypes (heteropolysaccharides). The molecular-weight of polysaccharidescan range into the millions of daltons. Specific examples ofcarbohydrates include glucose, galactose, xylose, fructose, sucrose, andglycogen. The term “simple sugar” typically refers to monosaccharides.

The term “lipid” generally refers to substances that are extractablefrom animal or plant cells by nonpolar solvents. Materials fallingwithin this category include the fatty acids, fats such as the mono-,di- and triacyl glycerides, phosphoglycerides, sphingolipids, waxes,terpenes and steroids. Lipids can also be combined with other classes ofmolecules to yield lipoproteins, lipoamino acids, lipopolysaccharidesand proteolipids.

“Fatty acids” generally refer to long chain hydrocarbons (e.g., 6 to 28carbon atoms) terminated at one end by a carboxylic acid group, althoughthe hydrocarbon chain can be as short as a few carbons long (e.g.,acetic acid, propionic acid, n-butyric acid). Most typically, thehydrocarbon chain is acyclic, unbranched and contains an even number ofcarbon atoms, although some naturally occurring fatty acids have an oddnumber of carbon atoms. Specific examples of fatty acids includecaprioic, lauric, myristic, palmitic, stearic and arachidic acids. Thehydrocarbon chain can be either saturated or unsaturated.

“Fats” are a particular class of lipids and are esters of fatty acidsand glycerol. Fats include mono-, di- and tri-acylglycerides.

A “nucleoside” is a compound of a sugar (typically a ribose ordeoxyribose) attached to a purine or pyrimidine base via an N-glycosyllinkage.

A “nucleotide” refers to a phosphate ester of pentose sugars in which anitrogenous base (typically a purine or pyrimidine base) is linked tothe C(1′) sugar residue. Most typically, a nucleotide is a nucleosideattached to a phosphoric group.

The term “steroid” refers to the large class of compounds that containthe tetracyclic cyclopenta[α]phenanthrene backbone that are part of themetabolism of an organism. A specific example is cholesterol.

The term “compound” or “component” refers to a molecule regardless ofmolecular weight found within an organism or cell. A compound orcomponent can be from the same class of compounds as a substrate ormetabolite.

An “organic acid” refers to any organic molecule having one or morecarboxylic acid groups. The organic acid can be of varying length andcan be saturated or unsaturated. Examples of organic acids include, butare not limited to, citric acid, pyruvic acid, succinic acid, malicacid, maleic acid, oxalacetic acid, and α-ketoglutaric acid. Organicacids can include other function groups in addition to the carboxylicacid group including, for example, hydroxyl, carbonyl and phosphate.

The term “naturally occurring” as used herein as applied to an objectrefers to the fact that an object can be found in nature. For example, apolypeptide or polynucleotide sequence that is present in an organism(including viruses) that can be isolated from a source in nature andwhich has not been intentionally modified by humans in the laboratory isnaturally occurring. Generally, the term naturally occurring refers toan object as present in a non-pathological (undiseased) individual, suchas would be typical for the species.

As used herein, “substantially pure” means an object species is thepredominant species present (i.e., on a molar basis it is more abundantthan other species in the composition, with the exception of solventspecies and metal ions), and preferably a substantially purifiedfraction is a composition wherein the object species comprises at leastabout 50 percent (on a molar basis) of all species present. Generally, asubstantially pure composition will comprise more than about 80 to 90percent of all species present in the composition. Most preferably, theobject species is purified to essential homogeneity (contaminant speciescannot be detected in the composition by conventional detection methods)wherein the composition consists essentially of a single species.

As used herein “normal blood” or “normal human blood” refers to bloodfrom a healthy human individual who does not have an active neoplasticdisease or other disorder of lymphocytic proliferation, or an identifiedpredisposition for developing a neoplastic disease. Similarly, “normalcells”, “normal cellular sample”, “normal tissue”, and “normal lymphnode” refers to the respective sample obtained from a healthy humanindividual who does not have an active neoplastic disease or otherlymphoproliferative disorder.

As used herein the term “physiological conditions” refers totemperature, pH, ionic strength, viscosity, and like biochemicalparameters which are compatible with a viable organism, and/or whichtypically exist intracellularly in a viable cultured yeast cell ormammalian cell. For example, the intracellular conditions in a yeastcell grown under typical laboratory culture conditions are physiologicalconditions. Suitable in vitro reaction conditions for in vitrotranscription cocktails are generally physiological conditions. Ingeneral, in vitro physiological conditions comprise 50-200 nM NaCl orKCl, pH 6.5-8.5, 20-45 C and 0.001-10 mM divalent cation (e.g., Mg⁺⁺,Ca⁺⁺); preferably about 150 mM NaCl or KCl, pH 7.2-7.6, 5 mM divalentcation, and often include 0.01-1.0 percent nonspecific protein (e.g.,BSA). A non-ionic detergent (Tween, NP-40, Triton X-100) can often bepresent, usually at about 0.001 to 2%, typically 0.05-0.2% (v/v).Particular aqueous conditions may be selected by the practitioneraccording to conventional methods. For general guidance, the followingbuffered aqueous conditions may be applicable: 10-250 mM NaCl, 5-50 mMTris HCl, pH 5-8, with optional addition of divalent cation(s) and/ormetal chelators and/or nonionic detergents and/or membrane fractionsand/or antifoam agents and/or scintillants.

The term “statistical correlation” refers to a statistical associationbetween two variables or parameters as measured by any statistical testincluding, for example, chi-squared analysis, ANOVA or multivariateanalysis. The correlation between one parameter (e.g., value for theflux of a metabolite) and a second parameter (e.g., disease state) isconsidered statistically significant if the probability of the resulthappening by chance (the P-value) is less than some predetermined level(e.g., 0.05). The term “statistically significant difference” refers toa statistical confidence level, P, that is <0.25, preferably <0.05, andmost preferably <0.01.

II. Overview

The present invention provides methods and apparatus for conductingmetabolic analyses, including methods for purifying metabolites ofinterest, screens to identify metabolites that are correlated withcertain diseases and diagnostic screens for identifying individualshaving, or being susceptible to, a disease.

Certain methods of the invention provide electrophoretic methods forseparating various metabolites using a plurality of electrophoreticmethods performed in series. Such separation methods can be utilized toconduct various metabolic analyses. For example, certain analyticalmethods of the invention involve administering a substrate labeled witha stable isotope to a subject. The isotopic composition or enrichment ofthe substrate prior to administration is known. After waiting a periodof time to permit the substrate to be utilized, a sample is withdrawnfrom the subject and used to determine the isotopic composition ofmultiple target analytes, the target analytes comprising the substrateand/or one or more target metabolites formed from the substrate.Typically, samples are obtained from the subject at different timepoints and the abundance of the isotope determined for the targetanalytes in each sample. In this way, the isotopic composition of thesubstrates can be measured as a function of time to allow a flux valuefor each of the target analytes to be determined. Various methods can beutilized to determine relative isotopic abundance of the isotope in thetarget analytes, including nuclear magnetic resonance spectroscopy,infrared spectroscopy and mass spectroscopy.

Unlike certain other methods that focus on the concentration of aparticular metabolite, certain methods of the invention are designed todetermine flux rather than a single concentration value. This simplifiesthe methods because flux values can be determined from the relativeabundance of the isotope label in the target analytes rather than havingto determine absolute concentration values. Furthermore, fluxdeterminations provide insight into certain biological processes thatare not observable from simple concentration determinations. Forexample, while concentration values may appear constant, flux canactually be changing. The concentration of any metabolite is determinedby the rates of all reactions involving the formation conversion andtransport of that metabolite. Therefore, increases in any two specificreactions (fluxes) involving both the formation and removal (conversionor transport) of the metabolite can yield the same apparentconcentration of the metabolite Flux can be altered in response to anumber of different stimuli, and thus can serve as sensitive indicatorof certain cellular states. For example, flux can be altered in responseto factors such as physiological state exposure to toxins andenvironmental insults, as well as various disease states such asinfection, cancer, inflammation and genetic based defects in metabolism.Thus, flux can be used to detect diverse cellular conditions or statesthat are not necessarily detectable by other methods.

In some methods of the invention, the samples obtained from the subjectare purified prior to determining the isotopic abundance of the isotopein the analytes. The purification procedure is used to at leastpartially remove other components in the cell from the target analytesof interest. Typically, this is accomplished by separating componentswithin the sample by multiple electrophoretic methods (i.e., multipledimensions) performed in series.

Certain methods combine the electrophoretic separation aspects of theinvention with certain mass spectroscopy techniques of the invention.Such arrangements enable relatively complex samples to be sufficientlyreduced in complexity so that samples containing a relatively limitednumber of target analytes can be directly injected into the massspectrometer to determine the isotopic abundance in the various targetanalytes of interest. Such systems can be automated to permit highthroughput analysis of metabolic samples.

The flux values determined for the various target analytes can be usedin a variety of different applications. For example, flux values forvarious subjects or various physiological conditions (e.g., diseased ornormal) can be used directly as inputs into a database. The flux valuescan also be employed in various screening applications. For example, theflux values from a test subject can be compared with corresponding fluxvalues for a diseased subject to identify potential markers for thedisease (i.e., metabolites that appear to be correlated with thedisease. Groups of flux values can be used to develop a “fingerprint”for different cellular states. Once a correlation between a diseasestate and one or more metabolites have been made, flux values for testsubjects can be compared with flux values for individuals havingdifferent diseases. Lack of a statistically significant differencebetween the test and diseased subjects indicates that the test subjecthas the disease or is susceptible to the disease. Changes in metabolicflux can be manifested as a change in the relative amounts ofalternative analytes produced from a single substrate at metabolicbranch points, and as the rates at which analytes resulting from serialconversions of a single substrate are produced.

III. Methods

A. General

By feeding a tissue, population of cells or an organism anisotopically-enriched substrate and following the ratio of isotopic tononisotopic metabolites in the cell over time, one can generate aquantitative picture of cellular metabolism. The relative metabolic fluxcan be ascertained by determining the ratio of the amount ofisotopically enriched analytes to normal analytes at any given timeusing a variety of different detectors capable of detecting the relativeabundance of different isotopes (e.g., mass spectrometry). At eachmetabolic branch point, the relative ratio of isotopic to nonisotopicproducts on each side of the branch point provides an indication of theflux of metabolite diverted into each branch of the metabolic pathway.Following the rate of change of the isotopic ratio in identifiablemetabolites along a linear metabolic pathway in pulse labeled culturesprovides an estimate of the metabolic flux through each step of thepathway. Metabolites become isotopically enriched in front of slowkinetic steps and remain isotopically poor immediately after thesesteps. Once specific changes in cellular metabolism, such as induced bytoxic challenge or infection, are identified using the techniquesdescribed herein, one can synthesize isotopically enriched compoundsthat can be used as specific diagnostic markers of these metabolicchanges, wherein the substrate is only metabolized or fails to bemetabolized in response to a specific disease state (see e.g., U.S. Pat.Nos. 4,830,010; 5,542,419; 6,010,846 and 5,924,995).

B. Administering Labeled Substrate to Subject

1. Types of Subject

A “subject” as used herein generally refers to any living organism fromwhich a sample is taken to conduct a metabolic analysis. Subjectsinclude, but are not limited to, microorganisms (e.g., viruses, bacteriayeast, molds and fungi), animals (e.g., cows, pigs, horses, sheep, dogsand cats), hominoids (e.g., humans, chimpanzees, and monkeys) andplants. The term includes transgenic and cloned species. The term alsoincludes cell or tissue cultures that can be cultured to carry on themetabolic process under investigation. The term “patient” refers to bothhuman and veterinary subjects.

If the subject is a population of cells or a cell culture, any of thestandard cell culture systems known in the art can be used. Examples ofsuitable cell types include, but are not limited to, mammalian cells(e.g., CHO, COS, MDCK, HeLa, HepG2 and BaF3 cells), bacterial cells(e.g., E. coli), and insect cells (e.g., Sf9). Further guidanceregarding cell cultures is provided in Sambrook et al. MOLECULARCLONING: A LABORATORY MANUAL, 2d ed. (1989) Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.

2. Types of Substrate

As used herein, the term “substrate” when used within the context of thechemical species being administered to a subject refers to any speciescapable of being metabolized by the subject of interest. Exemplarysubstrates include, but are not limited to, proteins, carbohydrates,amino acids, nucleotides, nucleosides, nucleic acids, fats, fatty acids,and steroids. A “metabolite” refers to a product derived from enzymaticconversion of a substrate administered to a subject, the conversionoccurring as part of a metabolic process of the subject. A “targetmetabolite” is a metabolite under study in an analysis (e.g., ametabolite for which a flux value is to be determined).

A substrate labeled with a stable isotope refers to a substrate having adistribution of stable isotopes significantly different from that foundin the corresponding naturally occurring substrate. The term stableisotope refers to isotopes of an element that are not radioactive. Theisotopes typically used in the methods of the invention include ¹³C, ²H,¹⁵N, ¹⁸O, and ³⁴S. Hence, when substrates are labeled with ¹³C, thesubstrate includes a mixture of carbon isotopes where the ¹³C isotope isincorporated in the substrate at an abundance level detectably greaterthan its natural abundance. For ¹³C detection by mass spectrometry, thislevel is 2-100%, and preferably 10-100% of all the C atoms present inthe substrate (i.e., the atoms in the substrate collectively have theindicated percentage enrichment). For substrates labeled with ¹⁵N, adetectable level is 0.75-100% ¹⁵N, and preferably 4-100% ¹⁵N. Forsubstrates labeled with ¹⁸O, a detectable level is 0.4-100% ¹⁸O, andpreferably 2-100% ¹⁸O. For compounds labeled with ³⁴S, a detectablelevel is 8.4-100% ³⁴S and preferably 42-100% ³⁴S. In some instances, thedesired abundance level is obtained by mixing substrates that areisotopically enriched with non-enriched substrate.

3. Amount of Substrate

The amount of substrate added varies depending upon several factors. Ingeneral, however, the substrate is added at a safe and effective amount.As used herein, the term “safe and effective amount” means that theamount of substrate is sufficient so that the isotopic abundance, or atleast a ratio of isotopic abundances, can be determined with thedetector of choice, but not so high so that the substrate causes undueadverse side effects. Thus, the amount administered should becommensurate with a reasonable risk/benefit ratio. For example, if thesubstrate is labeled with ¹³C, then the substrate is administered to thepatient in sufficient quantity such that the ¹³C/¹²C ratio in the targetanalyte(s) can be determined. However, the amount of substrateadministered is below the amount that causes undesired side effects(e.g., toxicity, irritation or allergic response). The safe andeffective amount depends upon factors such as the nature and amount ofthe sample acquired (e.g., gas or liquid and/or acquisition site), theweight of the test subject, and the relative concentration of theisotope in the substrate.

4. Mode of Administration

Typically a mixture of known amounts of both labeled and unlabeledsubstrate is administered to a subject. The mixture may contain between5-95% relative abundance of labeled substrate. More preferably, themixture contains between 25-75% labeled substrate. Most preferably, themixture contains about equimolar ratios of labeled and unlabeledsubstrate.

In certain methods, the substrate is administered to the subject as apulse. Pulsed additions or pulsed labeling refers to the timed additionof an isotopically labeled substrate, wherein the relative isotopicabundance of the isotopes is known. Long pulses can be used to estimatenet synthesis rates of particular biomolecules starting from the time ofthe pulse. In this instance, previous biomass contains no label, but newbiomass begins to accumulate the isotope in proportion to the isotopicabundance of the label in the substrate. If the pulse duration is longcompared to the turnover of the substrate and target analytes ofinterest, the net synthesis rate is measured. Short pulses(significantly shorter than the turnover rate) do not account fordegradation and recycle, so provide an estimate of the unidirectionalsynthesis rate.

The mode by which the substrate is administered to the subject can varybut should be administered in such a way that the substrate can bemetabolized within a reasonable time frame. The substrate can beadministered in substantially pure form or as part of a composition.Compositions can include pharmaceutically acceptable componentsincluding, but not limited to, diluents, emulsifiers, binders,lubricants, colorants, flavors and sweeteners, so long as thesecomponents do not interfere with the metabolism of the substrate beingadministered. Guidance on the incorporation of such optional componentsis discussed, for example, in The Theory and Practice of IndustrialPharmacy (L. Lachman, et al., Ed.) 1976; and Remington 's PharmaceuticalSciences, Mace Publishing Company, Philadelphia, Pa., 17th ed., (1985);and Langer, Science 249:1527-1533 (1990), each of which is incorporatedby reference in its entirety.

In some instances, the substrate is administered orally in solid form(e.g., solid tablet, capsule, powder, pill granule) or as part of aliquid solution (e.g., emulsion, suspension). When shaping into the formof tablets, as the carrier for the substrate, there can be usedexcipients such as lactose, sucrose, sodium chloride, glucose, urea,starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid,and potassium phosphate; binders such as water, ethanol, propanol,simple syrup, glucose solution, starch solution, gelatin solution,carboxymethyl cellulose, hydroxypropyl cellulose, methyl cellulose, andpolyvinyl pyrrolidone; disintegrators such as carboxymethyl cellulosesodium, carboxymethyl cellulose calcium, low-substitution degreehydroxypropyl cellulose, dried starch, sodium alginate, agar powder,laminaran powder, sodium bicarbonate, and calcium carbonate; surfactantssuch as polyoxyethylene sorbitan fatty acid ester, sodium laurylsulfate, and monoglyceride stearate; disintegration inhibitors such assucrose, stearin, cacao butter, and hydrogenated oil; absorptionaccelerators such as quaternary ammonium base, and sodium laurylsulfate; humectants such as glycerin and starch; absorbents such asstarch, lactose, kaolin, bentonite, and colloidal silicic acid; andlubricants such as purified talc, stearate, borax and polyethyleneglycol. Furthermore, tablets can be optionally formed into tabletssubjected to normal tablet coating, such as sugar coated tablets,gelatin coated tablets, enteric coated tablets, film coated tablets ordouble tablets and multilayer tablets.

When shaping into the form of pills, as the carrier for the substrate,there can be used excipients such as glucose, lactose, starch, cacaobutter, hardened vegetable oil, kaolin, and talc; binders such as gumarabic, tragacanth powder, gelatin, and ethanol; and disintegrators suchas laminarane and agar.

The substrate, alone or in combination with other suitable components,can also be made into aerosol formulations (i.e., they can be“nebulized”) to be administered via inhalation. Aerosol formulations canbe placed into pressurized acceptable propellants, such asdichlorodifluoromethane, propane, nitrogen.

Suitable formulations for rectal administration include, for example,suppositories, which consist of the packaged active ingredient with asuppository base. Suitable suppository bases include natural orsynthetic triglycerides, paraffin hydrocarbons, polyethylene glycol,cacao butter, higher alcohols, esters of higher alcohols, gelatin, andsemisynthetic glycerides. In addition, it is also possible to usegelatin rectal capsules that consist of a combination of the substratewith a base, including, for example, liquid triglycerides, polyethyleneglycols, and paraffin hydrocarbons.

Formulations of the substrate suitable for parenteral administration,such as, for example, by intraarticular (in the joints), intravenous,intramuscular, intradermal, intraperitoneal, and subcutaneous routes,include aqueous and non-aqueous, isotonic sterile injection solutions,which can contain antioxidants, buffers, bacteriostats, and solutes thatrender the formulation isotonic with the blood of the intendedrecipient, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives. In the practice of this invention, compositions canbe administered, for example, by intravenous infusion, orally,topically, intraperitoneally, intravesically or intrathecally. Thecompositions are formulated as sterile, substantially isotonic and infull compliance with all Good Manufacturing Practice (GMP) regulationsof the U.S. Food and Drug Administration.

When the substrate is administered to a population of cells, the cellsare typically suspended in a matrix containing the isotopically-enrichedsubstrate, The matrix is typically an aqueous solution and can alsocontain other nutrients. Depending upon the number of cells, the cellscan be suspended in standard culture flasks or within the wells of amicrotiter plate, for example.

C. Sample Collection

1. Sample Sources and Types

As noted above, the methods of the invention can be used to analyzemetabolism in essentially any living organism. The samples can come fromtissues or tissue homogenates, fluids of an organism or cells or cellcultures. Generally, samples are obtained from the body fluid of anorganism. Such fluids include, but are not limited to, whole blood,plasma, serum, semen, saliva, urine, sweat, spinal fluid, saliva,gastrointestinal fluids, sweat, cerebral fluid, and lacrimal fluids. Insome instances, samples are obtained from fecal material, buccal, skin,tissue biopsy or necropsy and hair. Samples can also be derived from exvivo cell cultures, including the growth medium, recombinant cells andcell components. In comparative studies to identify potential drug ordrug targets (see infra), one sample can be obtained from a diseasedsubject or cells and another sample a non-diseased subject or fromnon-diseased cells, for example.

2. Collection Options

Certain methods involve withdrawing a sample of blood from the subject.If whole blood is used, the sample typically is lysed by any of themethods known to those of skill in the art including, for example,freezing/thawing the sample. Urine can be collected by collecting theurine of the subject in a clean container. In some instances, a sampleis obtained from the breath of an individual (e.g., when the targetmetabolite is carbon dioxide). A variety of different devices andmethods have been developed to collect breath samples. For example, thebreath of a subject can be captured by having the subject inflate anexpandable collection bag (e.g., a balloon). The sample can then betransferred to a commercially available storage container for subsequentstorage and/or transport (e.g., the VACUTAINER manufactured byBecton-Dickenson Company). Other breath collection devices are describedin U.S. Pat. Nos. 5,924,995 and 5,140,993, which are incorporated byreference in their entirety. Tissue samples may be obtained by biopsy.

In the case of cell or tissue cultures, cells are collected bycentrifugation or filtration and then lysed according to standardprotocols (e.g., sonication, blending, pressurization, freeze thawingand denaturation). Alternatively, cells can be collected and lysed bythe addition of trichloroacetic acid (to a final concentration of 5-10%weight to volume), or similar use of other membrane lytic solvents(e.g., chloroform, diethyl ether, toluene, acetone, and ethanol). Suchmembrane lytic solvents can be used to precipitate macromolecularcomponents and selectively solubilize small molecule metabolites as aprecursor to subsequent electrophoretic separation techniques.

D. Target Analyte Separation

1. Preliminary Purification

Depending on the complexity of the sample (i.e., the number anddifferent types of components within the sample), the target analytes(i.e., substrate and/or target metabolites) are first at least partiallypurified from other components within the sample. If the sample containscellular debris or other material that might interfere with separation,such materials can be removed using any of a variety of known separationtechniques including, for example, forcibly extruding the sample throughsieve material, filtration, centrifugation (e.g., density gradientcentrifugation), and various chromatographic methods (e.g., gelfiltration, ion exchange or affinity chromatography).

Many macromolecules (e.g., proteins and nucleic acids) can be separatedfrom small molecules (e.g., nucleotides, acetyl CoA, mono- anddisaccharides, amino acids) by lysing the cells and quantitativelyprecipitating the macromolecules by treating the lysed cells with coldtrichloroacetic acid (e.g. 5-10% TCA weight to volume for 30 min onice), while most of the small molecules in the cell remain soluble.Additional separation methods are discussed, for example, by Hanson andPhillips (Hanson, R. S. and Phillips, J. A., In: Manual of methods forgeneral bacteriology, Gerhardt et al. (eds.)., Am. Soc. Microbiol.,Washington, D.C., p. 328 (1981)).

2. Multidimensional Electrophoresis

Once such initial purification steps have been completed (if necessary),the target analytes are typically further purified by conducting aplurality of electrophoretic methods conducted in series. For optimalperformance, samples whose ionic strength is particularly high can bedesalted using established techniques such as dialysis and dilution andreconcentration prior to conducting the electrophoretic methods. Themethods are said to be conducted in series because the sample(s)electrophoresed in each method are from solutions or fractionscontaining components electrophoresed in the preceding method, with theexception of the sample electrophoresed in the initial electrophoreticmethod. Each of the different electrophoretic methods is considered a“dimension”, hence the series constitutes an “multidimensional”separation.

The series of electrophoretic methods are typically conducted in such away that components in an injected sample for each electrophoreticmethod of the series are isolated or resolved physically, temporally orspacially to form a plurality of fractions, each of which include only asubset of components contained in the sample. Thus, a fraction refers toa solution containing a component or mixture of components that areresolved physically, temporally or spacially from other components in asample subjected to electrophoresis. Hence, resolved components canrefer to a single component or a mixture of components that areseparated from other components during an electrophoretic method. Asjust noted, samples in the various electrophoretic methods are obtainedfrom such fractions, with the exception of the first electrophoreticmethod in which the sample is the original sample containing all thecomponents to be separated.

Typically, these multiple electrophoretic methods in the series separatecomponents according to different characteristics. For example, onemethod can separate components on the basis of isoelectric points (e.g.,capillary isoelectric focusing electrophoresis), other methods canseparate components on the basis of their intrinsic or induced (throughthe application of a label to certain ionizable groups) charge-to-massratio at any given pH (e.g., capillary zone electrophoresis), whereasother methods separate according to the size of the components (e.g.,capillary gel electrophoresis).

Apparatus used to conduct various electrophoretic methods are known inthe art. In general, however, and as shown in FIG. 2A, the basicconfiguration of a typical capillary electrophoretic system utilized incertain methods of the invention includes a capillary 8 having two ends10, 12. One end 10 is in contact with an anode solution or anolyte 14contained in an anode reservoir 18 and the other end 12 is in contactwith a cathode solution or catholyte 16 in a cathode reservoir 20. Oneelectrode (the anode) 22 is positioned to be in electrical communicationwith the anode solution 14 and a second electrode 24 is positioned to bein electrical communication with the cathode solution 16. The cavity 26of the capillary 8 is filled with an electrophoretic medium, which insome instances can include a polymer matrix. As used herein the termanode refers to the positively charged electrode. Thus, negativelycharged species move through the electrophoretic medium toward theanode. The term cathode refers to the negatively charged electrode;positively charged species migrate toward this electrode. The anolyte isthe solution in which the anode is immersed and the catholyte is thesolution in which the cathode is immersed.

Sample is introduced into the capillary 8 via an inlet 28, and thecomponents therein resolved as an electrical field is applied betweenthe two electrodes 22, 24 by a power source 32 and the componentsseparate within the electrophoretic medium contained within theseparation cavity 26. Components can be controllably eluted from thecapillary via outlet 30 by controlling various parameters such aselectroosmotic flow (see infra) and/or by changing the composition ofone or both of the reservoir solutions (e.g., adjusting the pH or saltconcentration). Typically, the inlet 28 and the outlet 30 are simplyportions of the capillary formed to allow facile insertion into acontainer containing sample, anolyte or catholyte.

The term “capillary” as used in reference to the electrophoretic devicein which electrophoresis is carried out in the methods of the inventionis used for the sake of convenience. The term should not be construed tolimit the particular shape of the cavity or device in whichelectrophoresis is conducted. In particular, the cavity need not becylindrical in shape. The term “capillary” as used herein with regard toany electrophoretic method includes other shapes wherein the internaldimensions between at least one set of opposing faces are approximately2 to 1000 microns, and more typically 25 to 250 microns. An example of anon-tubular arrangement that can be used in certain methods of theinvention is the a Hele-Shaw flow cell (see, e.g., U.S. Pat. No.5,133,844; and Gupta, N. R. et al., J. Colloid Interface Sci.222:107-116 (2000). Further, the capillary, need not be linear; in someinstances, the capillary is wound into a spiral configuration, forexample.

An example of a system utilized with certain methods of the invention isillustrated in FIG. 1. This particular example shows a system in whichthree electrophoresis methods (initial, intermediate and final methods)are linked. The particular number of electrophoretic methods conductedcan vary, although the methods of the invention generally include atleast two electrophoretic methods. Most typically, the methods utilizetwo or three electrophoretic separation methods.

As can be seen in FIG. 1, an initial sample containing a plurality ofcomponents is introduced from sample container 50 into a firstseparation cavity of a first capillary 54 via sample inlet 52 utilizingany of a number of methods known in the art. Examples of suitablemethods include, pulling sample into the sample inlet 52 under vacuum(e.g., by pulling a vacuum on the sample outlet) or pushing sample intothe sample inlet 52 by pressurizing the sample container 50.Electromigration, often referred to as electrokinetic injection, isanother option. Once the initial sample is introduced into sample inlet52, the sample is then electrophoresed within the first separationcavity within the first capillary 54. The first separation cavitycontains a desired electrophoretic medium in which components in theinitial sample are at least partially resolved. Electrophoretic mediumcontaining resolved components is withdrawn from the first cavity,typically out the end of the separation cavity opposite the end in whichsample was introduced, although other withdrawal sites can be utilized(see infra). The withdrawn medium travels through outlet 56 and iscollected in separate containers 58 as multiple fractions. As shown inFIG. 1B, the containers 58 into which fractions are collected aretypically associated with a fraction collection device (a portion ofwhich is shown 60) capable of automatically advancing a set ofcontainers 58 to collect defined fractions (e.g., fractions of a certainvolume or covering a selected pH range).

A sample from a fraction collected from the first electrophoretic methodis then withdrawn from one of the plurality of containers 58, againutilizing techniques such as those described supra, via a second sampleinlet 62. Components in the sample from the fraction can then be furtherresolved by conducting an intermediate electrophoretic method (in theexample shown in FIG. 1, the second electrophoretic method). The sampleis introduced into a second capillary 64 via inlet 62 and the componentswithin the sample further separated within the electrophoretic mediumcontained within the second separation cavity of the second capillary 64and then eluted from the cavity via outlet 66. As with the firstelectrophoretic separation the electrophoretic medium containing theresolved or partially resolved components is collected as separatefractions within containers 68 typically aligned and advanced by asecond fraction collection device (a portion of which is shown 70).

A process similar to the second/intermediate method is conducted duringthe final electrophoretic method (the third electrophoretic separationmethod shown in FIG. 1). Sample is drawn via inlet 72 from a container68 containing a fraction obtained during the preceding method and isintroduced into a third or final electrophoretic cavity of a thirdcapillary 74 containing a third electrophoretic medium in whichcomponents contained in the applied sample are further separated byelectrophoresis. The third electrophoretic medium containing the furtherisolated proteins is subsequently withdrawn through outlet 76.

As noted above, more than the three electrophoretic methods shown inFIG. 1 can be performed. Such methods essentially involve repeating thegeneral steps described for the second/intermediate electrophoreticseparation above one or more times.

Following the final electrophoretic separation, a variety of differentoptions for analyzing the resolved components are available. As shown inFIG. 1, withdrawn electrophoretic medium can be passed through anoptional detector 78 in fluid communication with the separation cavityof the last capillary 74 to detect the resolved components (e.g.,labeled proteins). The detector 78, or an optional quantifying devicecapable of receiving a signal from the detector (not shown), can be usedto quantitate the amount of components within a certain portion orfraction of the electrophoretic medium. Detectors can also be utilizedto monitor the progress of separation after other columns as well.

Fractions are taken from the electrophoretic medium exiting the finalcapillary 74 or the detector 78 and analyzed by an analyzer 82 todetermine the molecular weight of the components within a fraction. Inparticular, the analyzer is used to determine the abundance of theenriched isotope in the target analytes. As described infra, a varietyof analyzers and techniques can be utilized to make this determination.For example, the analyzer 82 can be a mass spectrometer or an infraredspectrometer. Mass spectral data, for example, can be utilized todetermine the mass of the various components within a fraction. Theratio of labeled and unlabeled target analytes can be determined fromthe relative signal intensities for the labeled and unlabeled targetanalytes in the mass spectrum.

The specific elution conditions utilized to withdraw resolved componentsfrom the separation cavity depends upon the type of electrophoreticmethod conducted and is described more fully below for each of theelectrophoretic methods typically utilized in the present invention ingeneral, however, once components have been resolved within theseparation cavity, the conditions within the cavity are adjusted asnecessary (or the initial conditions selected) to achieve selective orcontrolled elution of the components from the cavity. For example,elution can be achieved by adding salts to, or adjusting the pH of, theanode or cathode solution, by regulating electroosmotic flow, byapplying hydrodynamic pressure or combinations of the foregoing.

Using the methods of the invention, resolved components can be isolatedphysically (e.g., placement into different containers such asillustrated in FIG. 1), spatially (e.g., spread throughout theelectrophoretic medium contained in the separation cavity) and/ortemporally (e.g., controlling elution so different components within asample elute from the capillary at different times). Thus, the methodsof the invention can separate mixtures of components as a function ofthe composition of elution buffers and/or time. The methods are notlimited to the spatial separation of components as are certaintraditional gel electrophoresis systems (e.g., 2-D gel electrophoresissystems for protein separation or pulsed-field and sequencing gelsystems for nucleic acid separations), or two-dimensional thin layerchromatography (2-D TLC) methods (for small molecule metaboliteseparations). Instead, with controlled elution, fractions can becollected so components within a fraction fall within a range ofisoelectric points and electrophoretic mobilities, for example.Controlled elution of components means that methods can be performed ina reproducible fashion. Such reproducibility is important in conductingcomparative studies and in diagnostic applications, for example.

During the elution or withdrawing of resolved components, generally onlya portion of the electrophoretic medium containing the resolvedcomponent is typically collected in any given fraction. This contrastswith certain 2-D methods in which a gel containing all the resolvedcomponents (e g., proteins) is extruded from the separation cavity andthe extruded gel containing all the components is used to conductanother electrophoretic separation. This also contrasts with certain 2-Dthin layer chromatography methods in which all the metabolites areseparated by their relative affinities for the matrix in a line usingone solvent system and are reseparated based on altered affinities by asecond solvent system applied perpendicularly to the direction of flowof the first solvent system.

Spacially, physically or temporally resolved components obtained at theconclusion of one electrophoretic method are then used as the source ofsamples for further separation of components contained within thefraction during a subsequent electrophoretic method. As illustrated inFIG. 1 typically samples from different resolved fractions aresequentially electrophoresed on the same capillary. Normally anothersample is not applied until the components in the preceding sample aresufficiently withdrawn from the separation cavity so that there is nooverlap of components contained in different fractions. Sequentialelution of fractions through the same column can significantly reduce oreliminate variations resulting from differences in cross-linking orelectric field strength that can be problematic in certain slab gelelectrophoretic methods. Hence, sequential separation can furtherenhance the reproducibility of the methods of the invention. Othermethods, however, can be performed in a parallel format, wherein samplesfrom different fractions are electrophoresed on separate capillaries.This approach allows for separations to be completed more quickly.However, the use of multiple capillaries can increase the variability inseparation conditions, thereby reducing to some extent reproducibilitybetween different samples.

In certain methods, the electrophoretic methods are conducted so thatpools containing similar components are obtained. For example, theelectrophoretic conditions can be controlled so that after the first orfirst few electrophoretic methods at least one pool containing primarilyrelated components is obtained (e.g., a pool containing primarilyproteins, polysaccharides, nucleic acids, amino acids, nucleotides,nucleosides, oligosaccharides, phosphorylated mono- or oligosaccharides,fats, fatty acids or organic acids). Pools of related components can beobtained by capitalizing on the distinctive feature of the differentclasses of components within a cell. For example, some classes ofcomponents are primarily singly charged (e.g., phosphorylated mono- oroligosaccharides), whereas others are primarily zwitterionic (e.g.,amino acids, proteins, nucleotides and some fats). CIEF can be used toresolve different zwitterionic components and can also be used toseparate zwitterionic species from non-zwitterionic species. Largecomponents (e.g., proteins) can be separated from smaller components(e.g., amino acids, mono- and disaccharides, nucleotides andnucleosides) using CGE. Through judicious selection of pH and bufferconditions, one can control the charge on various components and effectseparation of components having different charge-to-mass ratios by CZE.For example, certain buffers can be utilized that selectively complexwith certain components to introduce a desired charge to the selectedcomponents. An example of such a buffer is a borate buffer that can beused to complex to carbohydrates thereby imparting a negative charge tothe carbohydrates present in the sample. Additional details regardingthe electrophoretic methods are set forth infra.

By controlling the electrophoretic conditions to initially separate acomplex mixture into pools of different classes of components, one cansimplify an analysis considerably. For example, if the metabolite ofinterest is a carbohydrate, by controlling conditions appropriately sothat a pool of carbohydrates is obtained (e.g., using borate buffers),one can ignore fractions containing other classes of compounds. Thus,subsequent electrophoretic separations can simply be conducted with asample from the pool(s) of interest. Alternatively, if the pool ofsimilar compounds is sufficiently small, individual components of thepool can be completely resolved by mass spectrometric means after theelectrophoretic separations. Similarly, once conditions have beenestablished for a particular metabolite, it is not necessary to analyzeall fractions obtained from the various electrophoretic methods. Thereproducibility of the method enables a sample to be taken only from thefew fractions obtained adjacent the fraction(s) previously establishedto contain the target analytes of interest. Nonetheless, because certainmethods can be automated, even during initial screening tests, forexample, one can quickly analyze all the final fractions. Even scanningthe mass spectrum to identify signals for mass fragments of interest canbe automated through the use of computer programs to speed analysis.

E. Detection

Once the target analytes have been at least partially purified fromother molecules in the sample, the relative abundance of the isotope inthe unmetabolized substrate and/or target analytes is determined.Typically, this involves determining the ratio of the enriched isotopeto the more abundant isotope (e g., ¹²C/¹³C, ¹⁴N/¹⁵N, ¹⁶O/¹⁸O and³⁴S/³²S), although other measures of abundance can also be determined.

The measurement of the concentration of the enriched stable isotope canbe made according to a variety of options. One approach is to determinethe relative abundance of the isotopic label by mass spectrometry. Thetarget analytes generate distinct signals in the mass spectrum accordingto the mass to charge ratio of the substrate. The relative signalintensities for the different isotopic forms present enables therelative abundance of the different isotopic forms of each targetanalyte to be calculated, regardless of the absolute concentration ofthe analyte in the sample.

Methods for analyzing various biological molecules by mass spectrometryhave been established. Mass spectrometry can be used according to knownmethods to determine the masses of relatively small molecules (e.g.,nucleosides, nucleotides, mono and di-saccharides) as well as relativelylarge molecules. For example, mass spectrometry has increasingly beenapplied to protein identification. Electrospray and matrix assistedlaser desorption ionization (MALDI) are the most commonly used massspectrometric techniques applied to protein analysis because they arebest able to ionize large, low volatility molecular species.

In the case of DNA, the DNA can by hydrolyzed to deoxyribonucleosidesusing standard methods of hydrolysis. For example, the DNA can behydrolyzed enzymatically, such as for example with nucleases orphosphatases, or non-enzymatically with acids, bases or other methods ofchemical hydrolysis. Alternatively, intact DNA polymers can be analyzed.Deoxyribonucleosides can then be prepared for mass spectroscopicanalysis using standard techniques (e.g., synthesis of trimethylsilyl,methyl, acetyl and related derivatives or direct probe injection).

For the following major classes of metabolites, the following sourcesprovide additional guidance on mass spectral analysis of such moleculesand are incorporated by reference in their entirety: (1) lipids (see,e.g., Fenselau, C., “Mass Spectrometry for Characterization ofMicroorganisms”, ACS Symp. Ser., 541:1-7 (1994)); (2) volatilemetabolite (see, e.g., Lauritsen, F. R. and Lloyd, D., “Direct Detectionof Volatile Metabolites Produced by Microorganisms,” ACS Sympl Ser.,541:91-106 (1994)); (3) carbohydrates (see, e.g., Fox, A. and Black, G.E., “Identification and Detection of Carbohydrate Markers for Bacteria”,ACS Synp. Ser. 541: 107-131 (1994); (4) nucleic acids (see, e.g.,Edmonds, C. G., et al., “Ribonucleic acid modifications inmicroorganisms”, ACS Symp. Ser., 541:147-158 (1994); and (5) proteins(see, e.g., Vorm, O. et al., “Improved Resolution and Very HighSensitivity in MALDI TOF of Matrix Surfaces made by Fast Evaporation,”Anal. Chem. 66:3281-3287 (1994); and Vorm, O. and Mann, M., “ImprovedMass Accuracy in Matrix-Assisted Laser Desorption/IonizationTime-of-Flight Mass Spectrometry of Peptides”, J. Am. Soc. Mass.Spectrom. 5:955-958 (1994)). Further details regarding mass spectralanalysis is set forth infra.

Labeled carbon dioxide ([¹³C]CO₂) can also be detected using massspectrometry. Such approaches are described, for example, by Ewing, G.W., Instrumental Methods of Chemical Analysis, ⁴th ed., (1975); andKlein, P., et al., “Stable Isotopes and Mass Spectrometry in NutritionScience”, Analytical Chemistry Symposium Series 21:155-166 (1984), bothof which are incorporated by reference in their entirety.

An alternative to detection by mass spectrometry is to detect theisotope label using infrared (IR) spectroscopy or nuclear magneticresonance spectroscopy (NMR). Various target analytes can be detectedusing this approach, including carbon dioxide, for example. IR and NMRmethods for conducting isotopic analyses are discussed, for example, inU.S. Pat. No. 5,317,156; Klein, P. et al., J. Pediatric Gastroenterologyand Nutrition 4:9-19 (1985); Klein, P., et al., Analytical ChemistrySymposium Series 11:347-352 (1982); and Japanese Patent Publications No.61-42219 and 5-142146, all of which are incorporated by reference intheir entirety.

In certain methods, target analytes partially or completely purified bythe electrophoretic methods are subsequently transported directly to anappropriate director for analyzing the isotopic composition of thetarget analytes. In some methods, samples are withdrawn from theindividual fractions collected during the final electrophoreticseparation and injected directly onto a mass spectrometer to determinerelative abundances.

F. Flux Determination

In general, the flux of metabolites through each reaction step in anygiven pathway depends on the relative rates of the forward reaction andreverse reactions. As used herein, flux refers to the rate of change inconcentration of a target analyte as a function of time and sample size.The metabolic flux through any single metabolic conversions can bedetermined from the change in the relative abundance (RA_(t)) ofisotopically labeled analyte over time (t) according to the equation:

${Flux}_{analyte} = \frac{\ln\left\{ {1 - {{RA}_{t}/{RA}_{ss}}} \right.}{(t)\left( {{unit}\mspace{14mu}{of}\mspace{14mu}{sample}} \right)}$

where RA_(SS) is the relative abundance of the labeled metabolite atlong times. Relative abundance (RA) is the relative concentrations ofisotopically labeled substrate and/or target metabolite (i.e., thetarget analytes) determined from the ratio of the abundances of isotopiclabel in the target analytes. In some embodiments, the steady-staterelative abundance of the isotope can be considered equal to the knownratio in the initial substrate administered to the subject, such that aonly a single sample is needed to determine the metabolic flux. Inanother embodiment, the steady-state relative abundance of the isotopecan be predicted from simultaneous solution of the above equation fortwo or more relative abundance measurements taken from samples taken atdifferent time points. In another embodiment, the steady-state relativeabundance of the isotope can be measured directly from samples taken atlong times.

It is apparent to those skilled in the art that an alternative form ofthe above equation can be used to determine the flux of an analyte fromthe depletion of isotopically labeled analyte or substrate following areduction in the relative abundance of isotopically labeled substrate.This alternative form is:

${Flux}_{analyte} = \frac{\ln\left\{ \frac{\left( {{RA}_{t} - {RA}_{ss}} \right)}{\left( {{RA}_{o} - {RA}_{ss}} \right)} \right\}}{(t)\left( {{unit}\mspace{14mu}{of}\mspace{14mu}{sample}} \right)}$

where RA_(O) is the initial relative abundance of the isotopicallylabeled analyte prior to the administration of substrate to change therelative abundance. In one embodiment, RA_(O) is measured directly priorto administration of the new substrate. In another embodiment, RA_(O) isassumed to be the same as the relative isotope abundance in thesubstrate administered prior to the change.

The relative metabolic flux of substrate into any metabolic branch (i)in a network of n branched metabolic pathways is determined from theratio of relative abundances of iotopically labeled analyte appearing inanalytes downstream in each branch (j) of the metabolic pathway at anytime (t), but preferably at long times (i.e., at the steady-statecondition), according to the equation:

${Flux}_{branch}^{i} = {\frac{{RA}_{t}^{i}}{\sum\limits_{j = 1}^{n}\;{RA}_{t}^{j}}\left\lbrack {Flux}_{substrate} \right.}$

To determine flux, typically one or more samples are withdrawn from thesubject at different predetermined time points. The samples are thentreated, optionally purified, and then analyzed as described above todetermine one or more values for the relative concentration of theisotopic label in the target analytes at a sampling time(s) (t). Thesevalues can then be utilized in the formula set forth above to determinea flux rate for each of a plurality of target analytes. In someinstances, the target analytes used to determine flux are all organiccompounds (i.e., the analytes do not include carbon dioxide, forexample).

It is apparent to those skilled in the art that more accurate fluxdeterminations and standard errors of the estimated fluxes can also bemade using statistical curve fitting or parameter fitting methodsgenerally known in the art (e.g., Zar, J. H. Biostatistical Analysis,(Prentice-Hall, Englewood Cliffs, N.J., 1974)) and isotopic ratio dataobtained from a plurality of samples taken at different times.

The metabolic flux through a pathway depends on the rate determiningstep(s) within the pathway. Because these steps are slower thansubsequent steps in the pathway, a product of a rate determining step isremoved before it can equilibrate with reactant. Further guidance onflux and methods for its determination is provided, for example, byNewsholme, E. A. et al., Biochem. Soc. Stamp. 43:183-205 (1978);Newsholme, E. A., et al., Biochem. Soc. Symp. 41:61-110 (1976); andNewsholme, E. A., and Sart., C., Regulation in Metabolism, Chaps. 1 and3, Wiley-Interscience Press (1973).

IV. Synthesis of Labeled Substrates

The synthesis of isotopically labeled biological compounds has been wellestablished for a variety of different types of compounds including, forexample, nucleic acids, proteins, carbohydrates, as well as glycolysisand other metabolic pathway intermediates. Methods for isotopicallylabeling nucleic acids are discussed, for example, in U.S. Pat. No.6,010,846; the labeling of carbohydrates is discussed in U.S. Pat. No.4,656,133; and methods for labeling glycolysis intermediates isdiscussed in U.S. Pat. No. 5,439,803, all of which are incorporated byreference in their entirety.

In some instances, isotopically labeled biological compounds areobtained by feeding live organisms a diet enriched in one or more stableisotopes, harvesting and purifying the desired isotopically enrichedcompounds resulting from natural metabolism of the isotopically-enricheddiet. Alternatively, isotopically-enriched substrates can be synthesizedchemically from isotopically enriched precursors. Many suitablesubstrates for metomics studies (e.g., [¹³C]-fatty acids, [²H, ¹³C and¹⁵N]-amino acids, [¹³C and ²H]-peptides, and [¹³C and ¹⁵N]-nucleotides)are available from commercial sources such as Isotec (Miamisburg, Ohio),ICN Pharmaceuticals (Costa Mesa, Calif.), and Sigma-Aldrich (St. Louis,Mo.).

V. Capillary Electrophoresis Methods

A. Capillary Isoelectric Focusing Electrophoresis (CIEF)

1. General

Isoelectric focusing is an electrophoretic method in which zwitterionicsubstances such as proteins, nucleotides, amino acids and some fats areseparated on the basis of their isoelectric points (pI). The pI is thepH at which a zwitterionic species such as a protein has no net chargeand therefore does not move when subjected to an electric field. In thepresent invention, zwitterionic species can be separated within a pHgradient generated using ampholytes or other amphoteric substanceswithin an electric field. A cathode is located at the high pH side ofthe gradient and an anode is located at the low pH side of the gradient.

Zwitterionic species introduced into the gradient focus within the pHgradient according to their isoelectric points and then remain there.The focused components can then be selectively eluted as describedbelow. General methods for conducting CIEF are described, for example,by Kilar. F., “Isoelectric Focusing in Capillaries,” in CRC Handbook onCapillary Electrophoresis: A Practical Approach, CRC Press, Inc.,chapter 4, pp. 95-109 (1994); and Schwartz. H., and T. Pritchett,“Separation of Proteins and Peptides by Capillary Electrophoresis:Application to Analytical Biotechnology,” Part No. 266923(Beckman-Coulter, Fullerton, Calif., 1994); Wehr, T., Rodriquez-Diaz,R., and Zhu, M., “Capillary Electrophoresis of Proteins,” (MarcelDekker, N.Y., 1999), which are incorporated herein by reference in theirentirety.

2. System and Solutions

Because CIEF is primarily an equilibrium technique with low currentdensities, capillary heating typically is not a problem. Therefore,fairly large bore capillaries can be utilized. Suitable sizes include,but are not limited to, capillaries having internal diameters of 2-600μm, although more typically capillaries having internal diameters of25-250 μm are utilized. The use of relatively large bore capillariesmeans the method can use relatively high sample loads, which facilitatesdetection in subsequent dimensions. This feature of CIEF makes themethod well suited for the initial or one of the early electrophoreticseparations in the series. However, smaller diameter capillaries enabletemperature to be controlled more carefully and, in some methods, resultin improved signal detection (e.g., by laser induced fluorescence (LIF)detection of fluorescently labeled proteins).

The capillaries can have varying lengths. The length selected depends inpart on factors such as the extent of separation required. Typically,the capillaries are about 10 to 100 cm in length, although somewhatshorter and longer capillaries can be used. While longer capillariestypically result in better separations and improved resolution ofcomplex mixtures, longer capillaries also afford more opportunities forinteractions between species in the sample and the capillary wall andlower field strength. Consequently, there tends to be an upper limit oncapillary length beyond which resolution may be lost. Longer capillariescan be of particular use in resolving low abundance compounds. Furtherguidance on size and length of capillaries is set forth, for example, inPalmieri, R. and J. A. Nolan, “Protein capillary electrophoresis:Theoretical and experimental considerations for methods development,”in: CRC Handbook of Capillary Electrophoresis: A Practical Approach,Chp. 13, pgs. 325-368 (CRC Press, Boca Raton, 1994).

Generally, the capillaries are composed of fused silica, althoughplastic capillaries and PYREX (i.e., amorphous glass) can be utilized incertain methods. As noted above, the capillaries do not need to have around or tubular shape. Other shapes herein the internal dimensionbetween opposing faces is within the general range set forth in thissection can also be utilized.

A variety of different anode and cathode solutions can be used. Commonsolutions include sodium hydroxide as the catholyte and phosphoric acidas the anolyte. Similarly, a number of different ampholytes can beutilized to generate the pH gradient, including numerous commerciallyavailable ampholyte solutions (e.g., BioLyte, Pharmalyte and Servalyte).The selection of ampholytes and the breadth of the ampholyte gradientcan impact the resolution that is achieved by CIEF methods. Narrowampholyte gradients increase the number of theoretical plates in theseparation and can be beneficial for higher resolution separations overnarrow pI ranges.

CIEF methods utilized in the separations of the invention can beconducted in capillaries containing polymeric matrices or in freesolution (i.e., no gel or other polymeric matrix). Polymer matrices aretypically added to slow electroosmotic flow; however, in some instances,inclusion of polymeric matrices can restrict movement of larger proteins(Patton, W. F., “Defining protein targets for drug discovery usingProteomics,” paper presented at the IBC Proteomics conference, Coronado,Calif. (Jun. 11-12, 1998)). The use of free solutions is preferable insuch cases possibly in combination with other methods (e.g., capillarycoatings, gel plugs, or induced electric fields) to control theelectroosmotic flow.

3. Sample Preparation

In some instances, samples to be electrophoresed by CIEF are subjectedto denaturants to denature certain macromolecules, particularlyproteins. This ensures that the same components all have the same chargeand thus identical components focus at the same location rather thanpotentially at multiple zones within the capillary. Denaturants (e.g.,urea), non- and zwitterionic-surfactants (e.g., IGEPAL CA-630 or3-[{3-cholamidopropyl}dimethylammonio]-1-propane sulfonate) can also beused to suppress protein-wall and/or protein-protein interactions thatcan result in protein precipitation. An advantage of denaturing theproteins within a sample prior to electrophoresis is that the resultscan be used in comparisons with archival data typically obtained underdenaturing conditions.

A typical denaturing buffer includes urea and a nonionic or zwitterionicsurfactant as denaturants; a reducing agent (e.g., dithiothreitol (DTT)or mercaptoethanol) is typically included to reduce any disulfide bondspresent in the proteins. Other denaturants besides urea that can be usedinclude, but are not limited to, thiourea and dimethylformamide (DMF).Generally, guanidine hydrochloride is not utilized as a denaturantbecause of the very high ionic strength it imparts to a sample.Exemplary neutral detergents include polyoxyethylene ethers (“tritons”),such as nonaethylene glycol octylcyclohexyl ether (“TRITON” X-100),polyglycol ethers, particularly polyalkylene alkyl phenyl ethers, suchas nonaethylene glycol octylphenyl ether (“NONIDET” P-40 or IGEPALCA-630), polyoxyethylene sorbitan esters, such as polyoxyethylenesorbitan monolaurate (“TWEEN”-20), polyoxyethylene ethers, such aspolyoxyethylene lauryl ether (C₁₂E₂₃) (“BRIJ”-35), polyoxyethyleneesters, such as 21 stearyl ether (C₁₈E₂₃) (“BRIJ”721),N,N-bis[3-gluconamido-propyl]cholamide (“BIGCHAP”),decanoyl-N-methylglucamide, glucosides such as octylglucoside,3-[{3-cholamidopropyl}dimethylammonio]-1-propane sulfonate and the like.

The optimal amount of denaturant and detergent depends on the particulardetergent used. In general the denaturing sample buffers contain up to10 M urea (more typically 4-8 M and most typically 6-8 M). Specificexamples of suitable buffers (and denaturants and nonionic surfactantsfor inclusion therein) include those described by Hochstrasser etal.(Anal. Biochem. 173:424 (1988)) and O'Farrell (J. Biol. Chem.250:4007 (1975)). Denaturation is typically advanced by heating for 10min at 95° C. prior to injection into the capillary. Adjustments in thedenaturing sample buffers are made as necessary to account for anyelectroosmotic flow or heating effects that occur (see, e.g., Kilar, F.,“Isoelectric Focusing in Capillaries,” in CRC Handbook on CapillaryElectrophoresis: A Practical Approach, CRC Press, Inc., chapter 4, pp.95-109 (1994)).

The amount of sample injected can vary and, as noted above, depends inpart of the size of the capillary used. In general, the capillary isloaded with 0.1 to 5.0 mg of sample. Samples can be spiked with one ormore known pI standards to assess the performance of the method.

4. Elution

A variety of techniques can be utilized to elute or withdrawelectrophoretic medium containing resolved compounds out from thecapillary, but these methods fall into three general categories:hydrodynamic elution, electroelution and control of electroosmotic flow.

a. Hydrodynamic/Pressure Elution

Hydrodynamic or pressure elution involves applying pressure (or pullinga vacuum) via an appropriate pump connected with one end of thecapillary (see, e.g. Kilar, F., “Isoelectric Focusing in Capillaries,”in CRC Handbook on Capillary Electrophoresis: A Practical Approach, CRCPress, Inc., chapter 4, pp. 95-109 (1994)). However, hydrodynamicelution can cause band broadening and loss of resolution due to theparabolic flow profile that is formed in the capillary.

b. Electroelution

Electroelution, the other major approach, encompasses a variety oftechniques and in general involves altering the solution at the anodeand/or cathode to change some parameter (e.g., pH, ionic strength, saltconcentration) of the electrophoretic medium in the separation cavitysufficiently to effect elution.

i. Salt Mobilization

One electroelution approach involves addition of a salt to the catholyteor analyte, the salt having a non-acidic or non-basic counterion of thesame charge as the acidic or basic species within the reservoir to whichthe salt is added so that the counterion migrates from the reservoirinto the capillary. Since electrical neutrality must be maintainedwithin the capillary, the movement of the counterion into the capillaryresults in a reduction of the concentration of protons or hydroxidewithin the capillary and thus the pH is either raised or lowered. Thetheoretical basis for this type of mobilization is described by S.Hjerten, J. L. Liao. and K. Yao. J. Chromatogr., 387:127 (1987). Forexample, if the catholyte is sodium hydroxide (i.e., the basic speciesis hydroxide) then a salt having a negatively charged counterion otherthan hydroxide is added, for example sodium chloride. Movement ofchloride ion into the capillary reduces the local concentration ofhydroxide within the capillary, thereby decreasing the pH. As anotherexample, if the anolyte is phosphoric acid, then a salt having acounterion other than a proton is added, for example sodium phosphate.In this instance, movement of sodium ion into the capillary reduces thelocal concentration of protons within the capillary thereby increasingthe pH. As the pH is lowered or raised within regions of the capillarydue to the presence of the added counterion, elution occurs since theampholytes, and the focused components, migrate to the newly-defined pHregions corresponding to their isoelectric points. It has been shownthat both the type and concentration of salt used for mobilization hasimpact on the resolution of eluted compound peaks (see, e.g., R.Rodriguez-Diaz, M. Zhu, and T. Wehr, J. Chromatogr. A, 772:145 (1997)).For example, the addition of sodium tetraborate instead of sodiumchloride to the catholyte results in greatly increased resolution ofseparated proteins.

ii. pH Mobilization

Another technique, referred to herein as “pH mobilization” can also beutilized to elute compounds during CIEF. In this approach, an additiveis added to either the anode or cathode solution to alter the pH of thesolution. Unlike salt mobilization, however, the additive does notcontribute a mobile counterion that moves into the capillary. Here, theelution occurs as a result of the pH gradient being redefined by the pHof one or both of the reservoirs; therefore, components with pI's thatfall outside of this redefined pH gradient are eluted into either theanode or cathode reservoirs. Typically, the technique for cathodicmobilization proceeds as follows. Once the components are focused (e.g.,in a pI range of 3-10 using phosphoric acid as the anolyte and sodiumhydroxide as the catholyte) the cathodic capillary end is immersed intoa reservoir containing a solution that has a pH slightly less than 10,for example 50 mM imidazole (pKa 7) which has a pH of 9.85. Thecomponents are then allowed to refocus in the capillary, recognizable bya stabilization of the current through the capillary, the pI range nowbeing defined by 3-9.85. Any components with an isoelectric point of9.85 to 10 are eluted into the catholyte. The process can be repeatedwith catholyte containing a species that reduces the pH to slightly lessthan 9.85. In a stepwise fashion, the pH can continued to be reduced topH 7, thereby collecting separated components in fractions that span therange of 7-10. At this point, anodic mobilization can proceed byreplacing the anolyte with acids of increasing pKa to selectivelyincrease the pH from 3 to 7, thereby collecting fractions in the acidicrange (pH 3-7). The number of fractions can vary depending on thedesired fractionation resolution. Typically, these fractions are definedby differences of 0.05-0.5 pH units.

The technique of pH mobilization can be useful for samples containing ahigh concentration of one or more components (e.g., proteins) that maycause uneven spatial gradients inside the capillary. Using pHmobilization, only those components with isoelectric points below orabove the pI range that is defined by the reservoir pH's are eluted.This elution is, therefore, reproducible regardless of differences inthe shape of the capillary pH gradient or the presence of uneven spatialgradients inside the capillary.

c. Electroosmotic Flow (EOF)

Regulating the magnitude of electroosmotic flow (EOF) significantlyaffects the preceding electroelution methods (see supra) and is anothermeans by which resolved components can be selectively withdrawn uponconclusion of an isoelectric focusing separation. EOF is generated bythe ionization of silanol functionalities on the surface of a silicacapillary. Such ionization results in a layer of protons in theelectrophoretic medium at the surface of the silica capillary. Once anelectric field is applied, the layer of protons essentially constitutesa positively charged column of fluid that migrates toward the cathode,thereby causing bulk flow of the electrophoretic medium within thecapillary. Apparent velocity of components is equal to the sum of theelectroosmotic flow and their electrophoretic mobility. Thus, bycontrolling EOF, one can control or regulate the rate at whichcomponents move through the capillary. In CIEF methods, generally EOFshould be controlled to allow components within an injected samplesufficient time to focus before the proteins begin eluting from thecapillary.

A variety of techniques can be utilized to regulate EOF. One approachinvolves coating the walls of capillaries with various agents. Forexample, EOF alone glass silicate surfaces can be substantially reducedby silanizing them with a neutral silane reagent that masks asubstantial percentage of surface silanol groups (e.g., polyacrylamide,polyethylene glycol and polyethylene oxide). The magnitude of EOF can befurther controlled using silanizing reagents that include positively ornegatively charged groups. Positively charged coatings can be used tonullify surface negative charges to give a net surface charge of zero sothat EOF approaches zero. Coatings with higher positive charge densitiescan be used to reverse the direction of EOF for charged surfacematerials. This can be useful for slowing the net migration rates ofpositively charged sample species. Conversely, negatively chargedcoatings can be used to impart to or increase the magnitude of thenegative charge on surfaces, so as to increase the net migration ratesof negatively charged species. Representative positively chargedcoatings include trialkoxysilanes with polyethyleneimine, quaternizedpolyethyleneimine, poly(N-ethylaminoacrylamide) and chitosans, forexample. Representative negatively charged coatings includetrialkoxysilanes with carboxylate and sulfonate containing materialssuch as poly(methylglutamate) and 2-acrylamido-2-methylpropanesulfonatepolymers, for example. Charged coatings can also effectively reducesample adsorption, especially for samples having the same chargepolarity as the coating.

The separation medium can also include soluble agents for dynamicallycoating the walls of the separation cavity, to help reduce EOF duringelectrophoresis. Such soluble coating agents include quaternaryammonium-containing polymers, methyl cellulose derivatives, celluloseacetate, polyethylene oxide, chitosan, polyvinyl alcohol, polyethyleneglycol, polyethylenimine, and polyethylene oxide-polypropyleneoxide-polyethylene oxide triblock copolymers, for example. Typically,soluble coating agents are included at concentrations of about 0.05% toabout 4%, and more typically of about 1% to about 2%.

EOF and sample absorption can also be adjusted by including suitablereagents in the separation medium and running buffers. For example,negative surface charges can be masked by including a cationic additivein the medium, such as metal amine complexes, amines and polyamines suchas propylamine, triethylamine, tripropylamine, triethanolamine,putrescine, spermine, 1,3-diaminopropane, morpholine, and the like.Zwitterionic species comprising both negatively and positively chargedgroups that are isoelectric at the pH of electrophoresis can also beused, such as trialkylammonium propyl sulfonates, where alkyl is methyl,ethyl, propyl, and longer alkyl chains.

Another approach involves the generation of a current that opposes EOF.Typically, this is accomplished by applying a thin film of metal (e.g.iridium tin oxide or copper) to an external surface of the capillary.Application of current to the film generates a relatively small inducedcurrent within the capillary to reverse the EOF (see, e.g., Schasfoort,R. B. M., Schlautmann, S., Hendrikse, J., and van den Berg, A.,“Field-Effect Flow Control for Microfabricated Fluidic Networks,”Science, 286:942-945 (1999)).

Placing a porous plug at a location upstream from where sample isintroduced (upstream referring to a direction opposite the flow ofcomponents through the capillary) can also be utilized to control EOF.An example illustrating the location of the plug is illustrated in FIG.2B where the capillary 100 extends from the anode reservoir (not shown)at one end and the cathode reservoir at the other end (not shown).Component migration is in the direction of arrow 102 (i.e., from theanode to cathode direction).

As can be seen, the porous plug 104 is positioned to be upstream of thetrailing edge 106 of the sample once introduced into the capillary 100.The porous plug 104 is typically formed of a polymeric material andremains relatively stationary during electrophoretic runs. Examples ofsuitable materials from which the plug can be formed include polymerizedacrylamide with diacrylamide crosslinkers and agarose. Although notintending to be bound by any particular theory, the porous plug 104appears to function as a momentum transfer barrier by blockingreplacement of bulk fluid that in the absence of the plug 104 would movetoward the cathode reservoir.

In some methods, such as those containing large amounts of a particularcomponent (e.g., a protein) and/or a large number of differentcomponents, EOF should be reduced to very low levels to allow componentsthe opportunity to focus before the electrophoretic medium beginseluting from the capillary due to EOF. In certain methods an EOFof=0.5×10⁻⁶ cm²/V−s (at pH 8.6, and 25 ml4 TRIS-phosphate) has beenfound to allow ample time for the necessary focusing of proteins beforesample elutes from the capillary. Methods described above can reduceEOFs to these levels.

Thus, the foregoing approaches enable fractions to be collectedaccording to different criteria. Electroelution techniques, for example,can be used to collect fractions having a defined pH range. BOF elutionand pressure elution, in contrast, can be used to separate fractionsaccording to time of elution. Other techniques can also be utilized toelute resolved proteins after CIEF (see, e.g. Kilar, F., “IsoelectricFocusing in Capillaries,” in CRC Handbook or Capillary Electrophoresis:A Practical Approach, CRC Press, Inc.. chapter 4. pp. 95-109 (1994)).The controlled elution techniques are useful for enhancingreproducibility, an important factor in comparative and diagnosticmethods. Such techniques also provide improved tolerance of highabundance components such as proteins as compared to methods relying onspatial separation.

B. Capillary Zone Electrophoresis (CZE)

1. General

Capillary zone electrophoresis is an electrophoretic method conducted infree solution without a gel matrix and results in the separation ofcharged components (e.g., proteins, amino acids, fatty acids, fats,sugar phosphates, nucleic acids, nucleotides and nucleosides) based upontheir intrinsic charge-to-mass ratios. One advantage to CZE methods isthe ability to run with solvent systems that would normally beincompatible with typical water soluble gel matrices. Nonaqueous orwater miscible solvent systems can be used to improve the solubility ofhydrophobic and membrane bound components that would normally not beresolved by aqueous electrophoretic methods. General methods forconducting the method are described, for example, by McCormick, R. M.“Capillary Zone Electrophoresis of Peptides,” in CRC Handbook ofCapillary Electrophoresis: A Practical Approach, CRC Press Inc., chapter12, pp. 287-323 (1994); Jorgenson, J. W. and Lukacs, K. D., J. HighResolut. Chromatogr. Commun., 4:230 (1981); and Jorgenson, J. W. andLukacs, K. D., Anal. Chem. 53:1298 (1981)), each of which isincorporated by reference in its entirety.

2. System and Solutions

In general, the capillaries described above for CIEF are also suitablefor conducting CZE methods. Often the capillaries have internaldiameters of about 50 to 100 microns. Buffer composition and pH cansignificantly influence separations since separations in CZE are basedupon charge-to-mass ratios and the charge of a component is dependentupon the pH of the surrounding solution. At the extremes of pH (i.e.,below 2 and above 10) it is typically difficult to achieve resolution ofmany components because most charged groups on the components are eitherfully protonated or deprotonated and many components have a similarnumbers of acidic and basic residues per unit mass. Selectivity istypically enhanced at intermediate pH. For components having arelatively high percentage of acidic groups selectivity can often beenhanced near pH 4.5. For those components having a high concentrationof amine residues, selectivity can be enhanced near pH 10.

In CZE, solutions at the anode and cathode are typically the same. Thebuffer utilized can be essentially any buffer, the choice of bufferbeing controlled in part by the pH range at which the electrophoreticmethod is conducted and its influence on the detector noise. Examples ofuseful buffers at low pH include, but are not limited to, phosphate andcitrate; useful buffers at high pH include Tris/Tricine, borate and CAPS(3-(cyclohexylamino)-1-propane sulfonic acid). Further guidanceregarding suitable buffers and buffer additives is described byMcCormick, R. M. “Capillary Zone Electrophoresis of Peptides,” in CRCHandbook of Capillary Electrophoresis: A Practical Approach, CRC PressInc., chapter 12 , pp. 287-323 (1994).

In some instances, multiple CZE separations can be conducted withdifferent pH buffers to affect the fractionation of components. Forexample, a buffer of pH 3 can be used to resolve net positively chargedamine functional components (e.g., amino acids, and nucleosides), fromneutral (e.g., oligosaccharides, polysaccharides, simple sugars, andfatty acids), and from net negatively charged components (e.g.,phosphosugars and nucleotides). Fractions collected from this first CZEdimension can subsequently be further resolved in a second CZE dimensionat another pH. For example, the amino acids can be resolved fromnucleosides by a second CZE dimension conducted at pH 7 and fatty acidscan be resolved from the amino acids at pH of 5.5. The phosphosugars canbe further resolved from carboxylic acids by subsequent CZE separationat pH 11.

3. Elution

Elution can be accomplished utilizing some of the same methods describedabove for CIEF, namely pressure and EOF. As with CIEF, controlling EOFcan be important in certain methods to prevent electrophoretic mediumcontaining components from eluting from the capillary before thecomponents within the loaded sample have had an opportunity to separate.EOF can be controlled using the same methods utilized for controllingEOF in CIEF methods (e.g., coating the internal walls of the capillary,using a porous plug and generating an induced field to counteract EOF).Regulating and carefully selecting the pH and ionic strength of theelectrophoretic medium is another technique that can be used. BecauseEOF results from ionization of the silanol groups on the interiorcapillary surface, by conducting CZE at relatively low pH (e.g., pH 2-5,more typically about pH 3-4) the number of silanol groups that areionized is reduced. Such a reduction reduces EOF. To prevent sampleelution prior to complete separation, in certain analyses the EOF shouldbe reduced to <1×10⁻⁴ cm²/V−s (at pH 8.6 and 25 mM TRIS-phosphatebuffer). EOFs of this level can be obtained using the methods justdescribed.

Covalent modification of one or more analytes can also be usedstrategically as a means to control component elution. This techniqueinvolves adding a chemical moiety to certain components in the sample ora fraction collected from a CE step prior to injecting the sample intothe next capillary. By selecting modifying agents that preferentiallyreact with certain functional groups such as amino or carboxyl groups,the charge-to-mass ratio of certain components can be altered. Suchalterations can improve the resolution of components duringelectrophoresis as well as improve their detectability.

C. Capillary Gel Electrophoresis (CGE)

1. General

Capillary gel electrophoresis refers to separations of proteins, nucleicacids, or other macromolecules accomplished by sieving through a gelmatrix, resulting in separation according to size. In one format,proteins are denatured with sodium dodecyl sulfate (SDS) so that themass-to-charge ratio is determined by this anionic surfactant ratherthan the intrinsic mass-to-charge ratio of the protein (Cantor, C. R.and Schimmel, P. R. Biophysical Chemistry, W.H. Freeman & Co., N.Y.,(1980)). This means that proteins can be separated solely on the basisof size without charge factoring into the degree of separation. Theapplication of general SDS PAGE electrophoresis methods to capillaryelectrophoresis (CGE) is described, for example, by Hjerten, S.,Chromatogr. Rev., 9:122 (1967).

2. System and Solutions

The type of capillaries and their size are generally as described abovefor CZE. A variety of different buffers can be used, includingcommercially available buffers such as the “eCAP SDS” buffersmanufactured by Beckman (Hjertén, S., Chromatogr. Rev., 9:122 (1967);Beckman Instruments, “eCAP SDS 200: Fast, reproducible, quantitativeprotein analysis,” BR2511B, Beckman Instruments, Fullerton, Calif.,(1993); Gottlieb, M. and Chavko, M., Anal. Biochem., 165:33 (1987);Hochstrasser, D. F., et al., Anal Biochem., 173:424 (1988)). Variousbuffer additives can be utilized to increase resolution. Such additives,include, but are not limited to, small amounts of organic solvents, suchas N,N-dimethylformamide, cyclohexyldiethylamine, dimethoxytetraethyleneglycol and other polyols (e.g., ethylene glycol and polyethylene glycol)(see, e.g., Palmieri, R. and Nolan, J. A., “Protein capillaryelectrophoresis: Theoretical and experimental considerations for methodsdevelopment,” in: CRC Handbook of Capillary Electrophoresis: A PracticalApproach, Chp. 13, pgs. 325-368, CRC Press, Boca Raton, (1994); Wanders,B. J. and Everaerts, F. M., “Isotachophoresis in capillaryelectrophoresis,” in: CRC Handbook of Capillary Electrophoresis: APractical Approach, Chp. 5, pgs. 111-127, CRC Press, Boca Raton, Fla.,(1994)). The use of such solvents can improve the solubility of certaincompounds such as lipophyllic components in aqueous solution and enhancetheir stability against thermal denaturation, (Martinek, K., et al.,FEBS Lett., 51:152-155 (1975)) depress the electroosmotic flow in CZEand CGE (Altria, K. D. and Simpson, C. F., Anal. Proc., 23:453 (1986)),alter the electrical double-layer thickness at the capillary wall toinhibit protein binding interactions (Mc Cormick, R. M., “Capillary zoneelectrophoresis of peptides,” in: CRC Handbook of CapillaryElectrophoresis: A Practical Approach, Chp. 12, pgs. 287-323, CRC Press,Boca Raton, Fla., (1994)) and increase the viscosity of the runningbuffer which depresses the electroosmotic flow. Solvents utilized shouldbe compatible with the polymer matrix inside the capillary.

Isotachophoresis (IPE) can be used in certain methods to increaseresolution of charged components. For a general discussion of IPE. see,for example, B. J. Wanders and Everaerts, F. M., “Isotachophoresis inCapillary Electrophoresis,” in CRC Handbook of CapillaryElectrophoresis: A Practical Approach, chap. 5, pp. 111-127 (1994),which is incorporated by reference in its entirety. The velocity of acharged molecule moving through a capillary under a constant fieldstrength depends on its relative mobility which is a function of themass/charge of the molecule, temperature and viscosity of the mediumthrough which it is moving. However, in the absence of an adequateconcentration of highly mobile ions upstream of the sample ions, all theions eventually have to migrate at the speed of the slowest ion once theelectric field reaches a steady-state inside the capillary. Thiscondition causes the anions to stack in order of their relativemobilities at the interface of the leading and terminating buffers.

Under SDS denaturing conditions, all the components present in thesample have nearly identical mass/charges. By using a higher mass/chargeanion in the terminal buffer, one can force the components to move at aconstant slow speed through the capillary. This has two effects. First,components “stack” at the terminal edge of the leading buffer increasingtheir effective concentration inside the capillary. Second, anyseparation between components is based on their size. Therefore, the useof a hybrid IPE-CGE method in which the IPE is used for sample“stacking” can improve the resolution possible in the subsequent CGEseparation in some methods.

Various terminal buffer systems can be utilized in conjunction with IPEmethods. In one system, ε-aminocaproic acid (EACA) is used as theterminal electrolyte because it has a high mass/charge at high pH (>6).Tris(hydroxyethyl)aminomethane (TRIS) citrate at 0.05M is used as theleading buffer at pH=4.8 and as an intermediate stacking buffer atpH=6.5. The sample components (e.g., proteins) initially “stack” becauseEACA has a very low mobility in the pH 6.5 stacking buffer, but once theprotein “stack” and EACA reach the lower pH leading buffer, the mobilityof the EACA surpasses that of the components in the sample andseparation commences (see, e.g., Schwer, C. and Lottspeich, F., J.Chromatogr., 623:345 (1992)). This system can be used to create a hybridsingle column IPE-CPAGE system.

A 2 buffer system for IPE for the separation of proteins involvesdissolving sample in 0.01M acetic acid, which is also used as theterminal electrolyte. The leading and background buffer was 0.02Mtriethylamine-acetic acid solution at pH 4.4. The sample in terminalbuffer is sandwiched between the leading and background buffer. IPEcontinues until the background buffer overtakes the leading edge of theterminal buffer at which point IPE stops and separation begins (see,e.g.. Foret, F. et al., J.Chromatogr., 608:3 (1992)).

Another IPE approach that can be accomplished with any running buffer isto dissolve the sample in the running buffer but diluted to a lowerionic strength. This causes an increase in the electrical resistance inthe capillary where the sample plug is loaded and correspondingly fastermovement of the ions present in the sample matrix to running bufferboundary. The optimal ionic strength difference between the samplematrix and the running buffer is typically about 10-fold (see, e.g.,Shihabi, Z. K. and Garcia, L. L., “Effects of sample matrix onseparation by capillary electrophoresis,” in: CRC Handbook of CapillaryElectrophoresis: A Practical Approach, Chp. 20, pgs. 537-548, CRC Press.Boca Raton, Fla., (1994)).

3. Elution

In general, the discussion of elution for CZE applies to CGE. Elutioncan be accomplished utilizing pressure and EOF. As with CIEF and CZE,controlling EOF can be important in certain methods to preventelectrophoretic medium containing components from eluting before thecomponents within the applied sample have had an opportunity toseparate. The methods described supra for CIEF and CZE can be used tocontrol EOF at desired levels. To prevent sample elution prior tocomplete separation, in certain analyses the EOF should be reduced to<1×10⁻⁴ cm²/V−s (at pH 8.6 and 25 mM TRIS-phosphate buffer). EOF can bereduced to this range, for example, by controlling the pH of the buffer,by generation of a counteracting induced field, capillary coatings and aporous gel plug.

D. Detection Subsequent to Separation

As indicated in FIG. 1, electrophoretic solution withdrawn during thefinal electrophoretic separation can be directed toward an analyzer 82for the determination of the relative abundance of the labeled andunlabeled analytes. This arrangement provides considerable flexibilitywith regard to the nature of detection and does not limit the methods tothe standard absorbance and fluorescence techniques. The analyzer neednot be positioned to detect eluted components as shown in FIG. 1,however. In other arrangements, the analyzer is adapted so that it canscan resolved components within the separation cavity of the capillarytube itself. An example of such an arrangement involves the use of anear IR analyzer to detect ¹³C abundance in the resolved components.Since the metabolic flux is only determined from the relative abundanceof the isotopic ratios of the analytes of interest, any detector able toresole the relative abundance of labeled and unlabeled analytes can beused with the method. The quantitative sensitivity and accuracy of thedetector are relatively unimportant, since each analyte is presentsimultaneously in both the labeled and unlabeled forms. The detectorshould only be capable of precisely quantifying the relative abundanceof the isotope.

In some instances, a detector can be placed at the end of the finalcapillary column (and/or between other columns) to monitor flow and/orto determine when fractions should be collected.

E. Exemplary Systems

The methods of the invention are amenable to a variety of differentelectrophoretic methods. The controlled elution techniques wherebydefined fractions are separated spatially, physically or by time, andthe labeling and detection methods can be utilized in a number ofdifferent electrophoretic techniques. As noted above, the number ofelectrophoretic methods linked in series is typically at least two, butcan include multiple additional electrophoretic methods as well. In someinstances, each electrophoretic method in the series is different;whereas, in other instances certain electrophoretic methods are repeatedat different pH or separation matrix conditions.

Despite the general applicability of the methods, as noted above CIEF,CZE and CGE methods are specific examples of the type of electrophoreticmethods that can be utilized according to the methods of the invention.In certain methods, only two methods are performed. Examples of suchmethods include a method in which CIEF is performed first followed byCGE. Labeling, if performed, is typically performed after CIEF withdetection subsequent to elution of components from the CGE capillary. Inanother system, the first method is CGE and the final method is CZE.Isotope detection generally is not performed until the completion of thefinal electrophoretic separation. However, as indicated above, UV/VIS orLIF detection may be used during any or all separation dimensions tomonitor the progress of the separations particularly to determine whenfractions are to be collected. A third useful approach involvesconducting multiple CZE dimensions. These are specific examples ofsystems that can be utilized; it should be understood that the inventionis not limited to these particular systems. Other configurations andsystems can be developed using the techniques and approaches describedherein.

VI. Mass Spectroscopy Methods

Charged or ionizable analytes can be detected by a variety of massspectrometric methods. Certain method include electrospray (ESI) andmatrix assisted laser desorption ionization (MALDI) methods coupled withtime-of-flight (TOF) ion detection. ESI and MALDI are preferred becausethey are low energy ionization methods, generally resulting in lowfragmentation of most analytes, and are suitable for the ionization ofthe broadest possible array of target analytes. TOF detection is usefulbecause the accuracy of this technique in determining mass generallyallows isotopic resolution to the single atomic mass unit level, evenfor multiply charged species. However, other mass spectrometricionization and detection techniques can be usefully employed where theanalytes are particularly robust to fragmentation, the isotopicdifferences between labeled and unlabeled analytes is sufficientlylarge, and/or the number of charge states sufficiently low, to achieveresolution of the labeled and unlabeled analytes.

VII. Microfluidic Systems

A. Examples of configurations

In a variation of the electrophoresis systems described supra, thecapillaries are part of or formed within a substrate to form a part of amicrofluidic device that can be used to conduct the analyses of theinvention on a very small scale and with the need for only minimalquantities of sample. In these methods, physical fractions of samplestypically are not collected. Instead, resolved components are separatedspatially or by time. Methods for fabricating and moving samples withinmicrofluidic channels or capillaries and a variety of different designshave been discussed including, for example, U.S. Pat. Nos. 5,858,188;5,935,401; 6,007,690; 5,876,675; 6,001,231; and 5,976,336, all of whichare incorporated by reference in their entirety.

An example of a general system 150 that can be used with the methods ofthe present invention is depicted in FIG. 3A. The capillaries orchannels are typically formed or etched into a planar support orsubstrate. A separation capillary 152 extends from an anode reservoir154 containing anolyte to a cathode reservoir 156. The anode reservoir154 and the cathode reservoir 156 are in electrical contact with ananode and cathode 158, 160, respectively. A sample injection channel 162runs generally perpendicular to the separation capillary 152 and one endintersects at an injection site 164 slightly downstream of the anodereservoir 154. The other end of the sample injection capillary 162terminates at a sample reservoir 166, which is in electricalcommunication with a sample reservoir electrode 168. A detector 170 ispositioned to be in fluid communication with electrophoretic mediumpassing through the separation capillary 152 and is positioneddownstream of the sample injection site 164 and typically somewhatupstream of the cathode reservoir 156. In this particular configuration,fractions are withdrawn into the cathode reservoir 156. Movement ofelectrophoretic medium through the various channels is controlled byselectively applying a field via one or more of the electrodes 158, 160168. Application of a field to the electrodes controls the magnitude ofthe EOF within the various capillaries and hence flow through them.

An example of another configuration is illustrated in FIG. 3B. Thissystem 180 includes the elements described in the system shown in FIG.3A. However, in this arrangement, spacially or temporally resolvedfractions can be withdrawn at multiple different locations along theseparation capillary 152 via exit capillaries 172 a, 172 b and 172 c.Each of these capillaries includes a buffer reservoir 176 a, 176 b, 176c, respectively, and is in electrical communication with electrodes 174a, 174 b, 174 c, respectively. Movement of electrophoretic medium alongseparation capillary 152 and withdrawal of fractions therefrom into theexit capillaries 172 a, 172 b and 172 c can be controlled by controllingwhich electrodes along the separation capillary 152 and which of theexit capillary electrodes are activated. Alternatively, or in addition,various microfluidic valves can be positioned at the exit capillaries172 a, 172 b and 172 c to control flow. Typically, additional detectorsare positioned at the various exit capillaries 172 a, 172 b and 172 c todetect components in fractions withdrawn into these capillaries.

The configuration illustrated in FIG. 3B can be used in a number ofdifferent applications. One example of an application for which thistype of system is appropriate is a situation in which the type ofsamples being examined has been well characterized. If for example,certain fractions of components of interest have been previouslyestablished to fractionate at a particular location in the separationcapillary 152, then the exit capillaries 172 a, 172 b and 172 c can bepositioned at those locations to allow for selective removal of thecomponent(s) of interest.

In still another configuration, multiple exit capillaries branch fromthe end of the separation capillary 152 near the cathode reservoir 156,each exit capillary for withdrawing and transporting separate fractions.In this configuration also, withdrawal of fractionated components fromthe separation capillary can be controlled by regulating EOF within thevarious capillaries and/or by microfluidic valves.

Other components necessary for conducting an electrophoretic analysiscan be etched into the support, including for example the reservoirs,detectors and valves discussed supra.

B. Substrates

The substrate upon which the capillary or micro-channel network of theanalytical devices of the present invention are formed can be fabricatedfrom a wide variety of materials, including silicon, glass, fusedsilica, crystalline quartz, fused quartz and various plastics, and thelike. Other components of the device (e.g., detectors and microfluidicvalves) can be fabricated from their same or different materials,depending on the particular use of the device, economic concerns,solvent compatibility, optical clarity, mechanical strength and otherstructural concerns. Generally, the substrate is manufactured of anon-conductive material to allow relatively high electric fields to beapplied to electrokinetically transport the samples through the variouschannels.

In the case of polymeric substrates such as plastics, the substratematerials can be rigid, semi-rigid, or non-rigid, opaque, semi-opaque ortransparent, depending upon the use for which the material is intended.Plastics which have low surface charge when subjected to the electricfields of the present invention and thus which are of particular utilityinclude, for example, polymethylmethacrylate, polycarbonate,polyethylene terepthalate, polystyrene or styrene copolymers,polydimethylsiloxanes., polyurethane, polrinylchloride, polysulfone, andthe like.

Devices which include an optical or visual detector are generallyfabricated, at least in part from transparent materials to facilitatedetection of components within the separation channel by the detector.

C. Channel Structure/Formation

The size and shape of the channels or capillaries formed in thesubstrate of he present devices can have essentially any shape,including, but not limited to, semi-circular, cylindrical, rectangularand trapezoidal. The depth of the channels can vary, but tends to beapproximately 10 to 100 microns, and most typically is about 50 microns.The channels tend to be 20 to 200 microns wide.

Manufacturing of the channels and other elements formed in the surfaceof the substrate can be carried out by any number of microfabricatingtechniques that are known in the art. For example, lithographictechniques may be employed in fabricating glass or quartz substrates,for example, using established methods in the semiconductormanufacturing industries. Photolithographic masking, plasma or wetetching and other semiconductor processing technologies can be utilizedto create microscale elements in and on substrate surfaces.Alternatively, micromachining methods, such as laser drilling,micromilling and the like, can be utilized. Manufacturing techniques forpreparing channels and other elements in plastic have also beenestablished. These techniques include injection molding techniques,stamp molding methods, using for example, rolling stamps to producelarge sheets of microscale substrates, or polymer microcastingtechniques, wherein the substrate is polymerized within a micromachinedmold.

Further guidance regarding other designs and methods for using suchmicrofluidic devices such as described above can be found, for example,in U.S. Pat. Nos. 5,858,188; 5,935,401; 6,007,690; 5,876,675; 6,001,231;and 5,976,336, all of which are incorporated by reference in theirentirety.

VIII. Exemplars Utilities

The methods and apparatus of the invention can be used to separate anddetect a variety of different types of metabolic compounds, including,but not limited to, proteins, nucleic acids, polysaccharides, lipids,fatty acids, amino acids, nucleotides, nucleosides, monosaccharides anddisaccharides. Consequently, the methods and apparatus can be used in avariety of metabolic applications. For example, the methods can be usedto determine the flux of various metabolites. This capability can beused in biochemical, and especially metabolic, research in determininghow the flux of metabolites Wearies as a function of different cellularstates or in response to various external stimuli. The methods havevalue in clinical research by determining how the flux rates of variousmetabolites can vary between healthy and diseased states.

More specifically, the invention can be used to develop metomicdatabases. Such databases can include, for example, a register ofvarious metabolites detected for a particular state or physiologicalcondition of a subject. The database can be cross-referenced withadditional information regarding the subject and/or the metabolite. Forexample, concerning the subject, the database can include information onthe genus, species, age, race, sex, environmental exposure conditions,health status, sample collection methodology and type of sample. Fluxvalues can be included for each of the metabolites stored in thedatabase and can be cross indexed with metabolite concentration values,enzyme or transport protein concentration values responsible for themetabolic flux, or gene expression values corresponding to the proteinsresponsible for the metabolic flux.

Where the fluxes of a plurality of analytes are determined thatrepresent separable components of overall cellular metabolism, ametabolic fingerprint of the subject can be obtained. Analytes fromseparable components of the overall metabolism are functionally definedas compounds sufficiently separated by a series of enzymatic conversionsteps that the isotopic enrichment introduced by any single substratecan not be detected above the natural abundance of the isotope in thatanalyte, such that a second substrate must be introduced to measure theflux. In general, this functional criteria is satisfied if the targetanalyte is more than 5 conversion steps removed from the addedsubstrate. For example, the administration of labeled glucose as asubstrate is suitable for the determining the flux of severalphosphosugars in the glycolysis pathway. However, such administration isgenerally not sufficient to raise the relative abundance of ¹³C in aminoacids, fatty acids, and proteins because of the large number ofconversion steps separating the substrate from these target analytes. Insuch instances, the administration of an isotopically labeled amino acidcan be used to determine the flux of the amino acids and proteins; theadministration of an isotopically labeled fatty acid or acetate can beused to determine the metabolic flux of fatty acids.

In certain methods a plurality of metabolically separable substrates canbe administered simultaneously to a subject and a pluralist ofmetabolically separable target analytes detected from a single sampleobtained after a predetermined time from the subject. In a variation ofsuch methods, each of the metabolically separable substrates can belabeled with a different stable isotope. For example [¹⁸O]-glucose,[¹⁵N]-phenylalanine, and [¹³C]-acetate can be administeredsimultaneously to a subject to determine target analyte fluxes in theglycolysis, amino acid, and fatty acid metabolic pathways.

The invention can be employed in various screening applications. Forexample, the apparatus and methods of the invention can be used toidentify metabolites that are correlated with certain cellular states(e.g., certain diseases). For example, the methods can be utilized toidentify metabolites whose concentration or flux varies between healthyand diseased individuals or cells. Enzymes responsible for controllingthe concentration and flux of such metabolites are thus identified aspotential targets for drug therapy, for instance. In like manner,certain methods can be used to undertake toxicology studies to identifywhich metabolites, and thus the enzyme(s) controlling their formation,are affected by a toxic challenge.

Screening methods to correlate metabolites and certain cellular statesare similar to the general analytical methods set forth supra. Forinstance, a substrate labeled with a stable isotope is administered to atest subject having a disease and at least partially metabolized by thetest subject. Generally, one then partially or fully separates thetarget analytes of interest from other components in the sample underevaluation utilizing the various separation techniques described above.The relative abundance of the isotope in the target analytes isdetermined using a method capable of detecting the different isotopes todetermine a flux value for each of the target analytes in the testsubject. These determined values are then compared with thecorresponding flux values for a control that serves as a reference forflux values in a non-diseased state.

The control can be a value (e.g., an average or mean value) for acontrol subject(s) (i.e., someone without the disease) determined undersimilar conditions. Alternatively, the control can be a range of valuespreviously established to be representative of a non-diseased state. Adifference (e.g. a statistically significant difference) between fluxvalues for test and control indicates that the particular metabolite iscorrelated with the disease. Such a metabolite is a “marker” orpotential marker for the disease. The flux values for the controlsubject can be data obtained previously under like conditions to thetest, or the flux values can be determined for a control subjectundergoing simultaneous treatment with the test subject under identicalconditions.

Of course, similar screening methods can be conducted to developcorrelations between certain metabolites and cellular states other thandisease states. For example, methods can be conducted to identifymetabolites that are correlated with particular developmental stages,states resulting from exposure to certain environmental stimuli andstates associated with particular therapeutic treatments.

Multiple metabolites found to have a statistically significantdifference in flux values between diseased and control subjects (i.e.,markers) can be used to develop a “metabolic flux fingerprint” or simplya “fingerprint” for the disease. Such a fingerprint can subsequently beused to diagnosis the disease (see infra). Typically, such a fingerprintincludes at least 2, 3, 4, or 5 metabolites found to be correlated witha disease. In other instances, the fingerprint includes at least 6, 7,8, 9 or 10 such metabolites, and in still other instances 10, 15, or 20or more such metabolites.

The results from comparative studies are transferable to a variety ofdiagnostic applications. For example, the “marker” or “fingerprints” canbe used to screen or diagnose subjects to determine if they have, or aresusceptible to, a particular disease. The methods track those describedsupra, except that the substrate labeled with the isotope isadministered to a subject suspected to have the disease or susceptibleto it (or simply an interested individual seeking to determine if theyhave, or are susceptible to, the disease). Flux values for the testanalyte(s) (i.e., a “metabolic profile” for the test subject) are thancompared with reference flux values for individual test analytes(markers) or collections of markers (fingerprints).

The reference values to which the determined values are compared can berepresentative of either a healthy or diseased state. Furthermore, thereference value can be a particular value or a range of valuescorrelated with either a healthy or diseased state. For example, thereference can be a value (e.g., an average or mean value) for a controlsubject or subjects either having or not having the disease, thereference value determined under conditions similar to those under whichthe test subject was tested. Alternatively, the reference can be a rangeof values drawn from a population of control subjects either having ornot having the disease.

If the reference is for a normal or healthy state, a difference (e.g., astatistically significant difference) between flux values for testsubject and reference indicates that the test subject has, or is at riskof acquiring, the disease. Alternatively, lack of a difference indicatesthat the test subject does not have the disease and/or is at not at riskfor acquiring the disease. If, however, the reference is representativeof a diseased state, then a difference (e.g., a statisticallysignificant difference) between test and reference values indicates thatthe test subject does not have and/or is not at risk of acquiring thedisease. Conversely, lack of a indicates that the test subject eitherhas or is susceptible to acquiring the disease.

Diagnostic screens are not limited to simply detecting disease states.The screens can also be used to detect other types of cellular statessuch as certain developmental states or toxic states, for example.

When conducting such screening tests, typically the analysis can besimplified. For example, once markers for a disease have beenidentified, one can establish separation conditions such that thefraction(s) containing the markers or interest is(are) known. Thus,during the screening tests, only the components in those particularfractions need to be evaluated. The reproducibility of the separationand detection aspects of the invention facilitate such analyses.

Such screening methods can be conducted for a variety of differentdiseases. Diseases that can be evaluated with the methods of theinvention include, but are not limited to, various types of cancers,autism, microbial and viral infections, and various digestive disorders.

The methods of the invention have further utility in conductingstructure activity studies. For example, the methods can be used todetermine the effect that certain chemical agents or combination ofagents generally have on metabolism and, more specifically, the effecton the flux of certain metabolites of interest. Such tests can identifyagents that are disruptive to metabolism and pinpoint the particularmetabolites effected. In other applications, once an agent has beentested initially, the agent or combination of agents can be modified andthe analysis repeated to determine what effect, if any, themodifications had on metabolism. Such studies can be useful, forexample., in making derivatives of a lead compound identified duringinitial drug screening trials.

Metabolic engineering studies can also be conducted using the methods ofthe invention. In such studies, a gene involved in metabolism can begenetically engineered to include certain desired mutations, or thepromoter of a gene can be genetically engineered to increase or decreasethe relative expression level of the gene. Using the methods describedherein, one can determine what effect, if any, the geneticallyengineered changes have on the metabolism of the test subject.

The following examples are offered to illustrate, but not to limit, theclaimed invention.

EXAMPLE 1 CZE Separation of Unlabeled Proteins

Each of five proteins (see Table 1) were obtained from Sigma-Aldrich andwere suspended at 5 mg/ml in an aqueous denaturing sample bufferconsisting of 25 MM tris(hydroxymethyl)aminomethane phosphate (pH 4.0),0.5% by weight IGEPAL CA-630 (obtained from Sigma-Aldrich, Cat # I3021),and 1% by weight tris(2-carboxyethylphosphine)hydrochloride (TCEP,obtained from Pierce, Cat #20490ZZ). The protein samples were denaturedin this sample buffer by heating at 95° C. for 15 min. Each of the fivedenatured protein samples were diluted into a cZE sample buffer tocreate a final solution consisting of 25 mMtris(hydroxymethyl)aminomethane phosphate buffer (pH 4.0), 8 M Urea, anda final concentration of 0.2 mg/ml of each of the five proteins. Controlsamples were also prepared of each denatured protein separately at 0.5mg/ml final concentration in the same sample buffer.

TABLE 1 Protein Standards Protein Cat # pl MW (kDa) Hen egg whiteconalbumin C 0755 6.0, 6.3, 6.6 76.0 Bovine serum albumin B 4287 5.4,5.5, 5.6 66.2 Carbonic anhydrase II T 6522 4.5 21.5 Rabbit muscle GAPDHG 2267 8.3, 8.5 36.0 Bovine ribonuclease A R 5503 9.6 13.7

The mixed protein sample and each of the control samples were run by CZEin a 60 cm×75 μm fused silica capillary (Beckman Coulter). An 800 μmdetection window was located 50 cm from the anodic end of the capillary.A 160 nl sample volume was pressure injected at the anodic end and theseparations conducted at 500 V/cm in a 25 mM TRIS-phosphate and 8 M urearunning buffer at pH 4.0. Protein detection was accomplished by UVadsorption at 214 nm.

The individual unlabeled proteins were not resolved under theseconditions (see FIG. 4). The electrophoretic mobility of each proteinwas determined from replicate runs of the individual protein controls(FIG. 5) and correlated with the predicted mass to charge ratio of theproteins at pH 4.0 (FIG. 6). The mass to charge ratio for each of theunlabeled proteins was determined from the published protein sequencesobtained through Genbank in the manner described by Canter, C. R. andSchimmel, P. R., Biophysical Chemistry, W.H. Freeman and Co., New York,(1980), which is incorporated by reference in its entirety.

EXAMPLE 2 CZE Separation of Labeled Proteins, with Fraction Collection

Each of the five proteins described in Example 1 was suspended at 10mg/ml in a denaturing buffer containing 1% by weight of sodium dodecylsulfate and 1% by volume 2-mercaptoethanol.. The proteins were denaturedin this buffer by heating at 95° C. for 15 min. The denatured proteinsamples were labeled with 4-sulfophenylisothiocyanate (SPITC) obtainedfrom Sigma-Aldrich (Cat #85,782-3) and used as supplied. Labeling wasaccomplished by adding 0.01 ml of triethylamine, 0.01 ml of 2 M aceticacid and 0.02 ml of a 10% bad weight solution of SPITC in water to 0.1ml of each denatured protein sample. The reaction mixture was heated at50° C. for 24 h.

A quantity of 0.05 ml of each of the SPITC-labeled protein standards wasmixed together and separated by cZE as described in Example 1, with theexception that the pH of the separation buffer was adjusted to 3.0. Theindividual SPITC-labeled proteins were resolved (FIG. 7). Thus, thisexample taken in view of the results for Example 1 in which unlabeledproteins were poorly resolved demonstrates the positive effect thatlabeling can have when done prior to a cZE separation. Fractions werecollected by electroelution into separate vials containing theseparation buffer at the times indicated. The identities of theSPITC-labeled proteins were determined by subsequent cGE analysis of thefractions.

EXAMPLE 3 CIEF First Dimension Separation with Fraction Collection

Bovine Serum Albumin, Carbonic Anhydrase, and Conalbumin were used assupplied from Sigma-Aldrich (Table 1). Each protein was denatured asdescribed in Example 1. A 0.01 ml aliquot of each denatured proteinsample was added to 0.2 ml of the CIEF focusing buffer. The CIEFfocusing buffer consisted of 0.4% by weight hydroxymethyl cellulosesolution (Beckman-Coulter eCAP CIEF Gel Buffer, Cat #477497) containing1% by volume pH 3-10 Ampholytes (Fluka, Cat #10043) and 1% by weight3-[(3-cholamidopropyl)dimethylarnnonio]-1-propane sulfonate.

A poly(ethylene glycol)-coated 60 cm long 0.1 mm internal diameter fusedsilica capillary (Supelcowax 10, Supelco, Cat #25025-U) was filled withthe protein sample in the focusing buffer. The capillary contents werefocused between 10 mM phosphoric acid and 20 mM NaOH reservoirs for 7.5min at 500 V/cm and 25 C. A 0.5 psi pressure gradient was then appliedbetween the anolyte and catholyte reservoirs to facilitate the elutionof the focused proteins in the direction of the electroosmotic flow.

The protein peaks were detected by monitoring the ultraviolet absorptionat 214 nm through an optical window in the capillary positioned 50 cmfrom the low pH end. The current through the capillary was alsomonitored (FIG. 8). Fractions (B-G) were collected into 0.05 ml of 20 mMNaOH contained in separate reservoir vials for the times depicted (FIG.8). Only fractions F and G were found to contain protein (see Example4). Fraction G was found to contain carbonic anhydrase and no conalbuminor bovine serum albumin. Conalbumin and bovine serum albumin were foundto coelute in the peak observed in fraction F. This experimentillustrates the partial separation of a mixture of proteins in a singledimension. Further resolution was achieved in the second dimension (seeExample 4).

EXAMPLE 4 CGE Second Dimension Separation of CIEF Fractions

Each of the CIEF fractions (B-G) collected during the CIEF separationdescribed in Example 3 were evaporated in a Savant Model SC210A Spin-Vapto a final volume of 0.005 ml to concentrate any protein present in thefraction. A quantity 0.01 ml of SDS sample buffer was added to eachprotein concentrate. The SDS sample buffer consisted of 0.1 ml of eCAPSDS sample buffer (Beckman Coulter, Cat #241525), 0.01 ml of eCAP OrangeG Reference Marker (Beckman Coulter, Cat #241524), and 0.09 ml ofanhydrous glycerol.

Each sample was then run in CGE mode using a linearpoly(acrylamide)-coated fused silica capillary 60 cm long with a 100 μminternal diameter. The eCAP SDS 14-200 Gel buffer (Beckman-Coulter Cat#477416) was used for the separation and in both reservoirs. Theseparation was conducted at 20° C. and 500 V/cm for 50 min. Ultravioletdetection of the proteins was accomplished at 214 nm through an opticalwindow positioned 50 cm from the sample injection end of the capillary.Molecular weight calibration was conducted in a separate run using eCAP.MW Standards (Beckman-Coulter Cat #477418) as described by themanufacturer. A 100 sec sample injection at 0.5 psi was used to loadeach sample into the capillary.

The resulting electropherograms showed no detectable protein in any cIEFfraction except fractions F (FIG. 9) and G (FIG. 10). The molecularweight of the two proteins seen in fraction F (FIG. 9) correspond tothat of bovine serum albumin and conalbumin (Table 2). The molecularweight of the protein seen in fraction G (FIG. 10) corresponded to thatof carbonic anhydrase (Table 1). It is observed that the second cGEdimension was necessary to fully resolve bovine serum albumin fromconalbumin, which were not resolved by a single cIEF mode (Example 3).

EXAMPLE 5 Role of Metabolism in Autism

The methods of the invention can be utilized to ascertain whethervarious diseases have a metabolic basis and even to pinpoint themetabolic basis. As a specific example, the methods and apparatus of theinvention can be used to establish whether autism (or the severity ofautistic symptoms) results from dietary factors, determine the mechanismthrough which diet may influence autism, and establish a simple ¹³Cmetabolic assay to facilitate diagnosis of such dietary influences.

Elevated exogenous peptides have been found in the blood, urine, andcerebral spinal fluids of large numbers of autistic children (Reichelt,K. et al., J. Appl. Nutrition, 42:1-11 (1990); Reichelt, K. et al.,Developmental Brain Dysfunction, 7:71-85 (1994); Reichelt, K. et al.,Brain Dysfunction, 4:308-319 (1991); Gillberg, C., “The role ofendogenous opioids in autism and possible relationships to clinicalfeatures,” in Wing, L. (ed.), Aspects of Autism: Biological Research,pp. 31-37, Gaskell, London, (1988); Shattock, P., A., et al., BrainDysfunction, 3:328-345 (1990)). Precedents from celiac disease, and anumber of clinical case studies documenting significant patientimprovements on grain and dairy dietary restrictions, have led to threehypotheses regarding dietary effects on autism. (See, e.g., Fukudome, S.and Yoshikawa, M., FEBS Lett., 296:107-111 (1992); Fukudome, S. andYoshikawa, M., FEBS Lett. 316:17-19 (1993); Reichelt, K. et al., J.Appl. Nutrition, 42:1-11 (1990); Reichelt, K. et al., DevelopmentalBrain Dysfunction, 7:71-85 (1994); Reichelt, K. et al., BrainDysfunction, 4:308-319 (1991); Shattock, P., A., et al., BrainDysfunction, 3:328-345 (1990); Lewis, L. S., “Dietary intervention forthe treatment of autism: Why implement a gluten and casein free diet?,”in: Biological Treatments for, Autism and PDD, pp 196-226, (Sham, 1998);Serousi, K., “Following a different road. A child's documented recoveryfrom autism,” in: Biological Treatments for Autism and PDD, pp 265-289,(Shaw,1998)).

The first theory, suggested by Lewis, is that a mild form of celiacdisease may exist in many, if not all, autistic children, leading topeptide malabsorption from the gut and associated neurological symptoms(Lewis, L. S., “Dietary intervention for the treatment of autism: Whyimplement a gluten and casein free diet?,” in: Biological Treatments forAutism and PDD, pp 196-226, (Shaw, 1998)). Shaw has proposed a secondtheory, namely that the exogenous peptides observed are a consequence ofintestinal yeast infections, which are stimulated by the carbohydratecontent of grains and milk (Shaw, W., et al., “Increased excretion ofanalogs of Krebs cycle metabolites and arabinose in two brothers withautistic features,” Clin. Chem., 41:1094-1104 (1995); and Shaw, W.,“Organic acid testing, byproducts of yeast and their relationship toautism,” in: Biological Treatments for Autism and PDD, pp. 31-65, (Shaw,1998)). Intestinal yeast may directly produce exogenous peptides assecondary metabolites or the peptides may form in the blood indirectlythrough crosslinking caused by the high levels of reducing sugars theyproduce. Based on clinical data concerning the efficacy of secretin andpeptidase supplementation in the diet of autistic children, Shaw hasproposed a third possible mechanism, suggesting that digestive enzymesmay not be functioning properly in autistic patients (Shaw, W.,“Abnormalities of the digestive system,” in: Biological Treatments forAutism and PDD, pp. 124-138, (Shaw, 1998)).

How the methods of the invention can be used to distinguish betweenthese theories can be seen from a two compartment pharmacokinetic modelfor peptide absorption through the gut (Notari, R. E., Biopharmaceuticsand Pharmacokinetics: An Introduction, 2^(nd) ed., Marcei Dekker, N.Y.,(1975)). The flux of exogenous peptides through the intestinal wall(g_(p)) can be represented by:g _(p) =k _(t)(K _(a) C _(p) ^(i) −K _(b) C _(p) ^(b)   (1)where k_(t) is the mass transport coefficient for the intestinal wall,K_(i) and K_(b) are the equilibrium constants for dissolution of thepeptide in the intestinal wall and its concentrations in theintestine(C_(p) ^(i)) and blood C_(p) ^(b) respectively.

The flux of peptides through the intestinal wall competes with the rateof peptidase digestion of the peptide in the gut (r_(p)), which werepresent by:

$\begin{matrix}{r_{p} = \frac{{kE}_{p}C_{p}^{i}}{\left( {K_{m} + C_{p}^{i}} \right.}} & (2)\end{matrix}$where k is the rate constant, Ep is the peptidase concentration in thegut and K_(m) is the Michaelis constant.

Since typically C_(p) ^(i) >>K_(m), the peptide concentration in theblood as a function of time (t) is given by:

$\begin{matrix}{C_{p}^{b} = {\frac{C_{p_{o}}^{i}}{\frac{{kE}_{p}V_{i}}{k_{t}{SC}_{p_{o}}^{i}} - \left( \frac{K_{b}}{K_{i}} \right) + \left( \frac{V_{b}}{V_{i}} \right)}\left\{ {{\mathbb{e}}^{{- k_{t}}{S{\lbrack{{(\frac{K_{b}}{V_{b}})} - {(\frac{K_{i}}{V_{i}})}}\rbrack}}t} - {\mathbb{e}}^{{- \frac{kE}{C_{p_{o}}^{i}}}t}} \right\}}} & (3)\end{matrix}$where V_(i) is the volumes contained by the intestine, V_(b) is thedistribution volume in the body, and C _(p) _(O) ^(i) is the initialconcentration of the peptide bolus in the gut. Where K_(i)≈K_(b) andV_(i)≈V_(b), equation 3 can be approximated by:

$\begin{matrix}{C_{p}^{b} = {\frac{k_{t}{SC}_{p_{o}}^{i}}{{kEV}_{i}}\left\lbrack {1 - {\mathbb{e}}^{{- {(\frac{kE}{C_{p_{o}}^{i}})}}t}} \right\rbrack}} & (4)\end{matrix}$From equation 4 it is readily apparent that the amount of peptidecrossing the intestinal wall is determined by the ratio of thepermeation rate (k_(t)S) to the rate of peptide digestion in the gut(kEV_(i)).

Equally important is the rate of accumulation of peptide in the blood,which is predicted (equation 4) to depend primarily on peptidaseactivity in the gut (kE). Therefore, measurements of the time course ofpeptide accumulation in the blood can be used to identify the underlyingmechanism driving maladsorption and provide significant insights for themost appropriate course of therapy. More specifically, this provides thebasis for distinguishing between the two possible reasons for theappearance of exogenous peptides in the blood and urine of autisticchildren, namely: (1) the intestinal wall is compromised as in celiacdisease, giving rise to increased peptide permeability (k_(t)), or (2)autistic children may suffer from peptidase deficiency (erg., lowpeptidase enzyme levels or lower than normal peptidase activity ).

To identify the source of exogenous peptides arising from wheat (i.e.,improper digestion and malabsorption or microbial stimulation) autisticchildren (and t control group) are fed a mixture of ¹³C-enriched andnormal wheat flour. Exogenous peptides are identified from blood plasmaand urine of autistic and control groups. Mass spectrometric techniquesare used to establish the stable isotope ratio of any exogenous peptidesidentified. Peptides exhibiting two predominant isotopic peaks withrelative abundances equal to that of the ingested flour mixture can onlybe derived directly from the wheat proteins themselves. Peptides thatexhibit a multitude of isotopic peaks in relative abundances that varyfrom that of the ingested flour mixture, can only have been formed aftercomplete digestion of the wheat proteins (i.e., after the constituent¹³C and ¹²C amino acids had a chance to intermingle). In a follow upexperiment patients are fed a flour mixture predigested with proteases.These patients should not exhibit any exogenous peptides derived fromwheat proteins but should still exhibit peptides derived from microbialor disease-related synthesis within the body.

Related methods are used to identify if any of the exogenous peptidespass the blood brain barrier (have the potential to cause neurologicaldisorders) and identify their ultimate source. Peptides derived directlyfrom incomplete digestion and malabsorption of food proteins can bedetected directly in cerebral spinal fluid samples taken from autisticchildren after ingestion of the flour mixture according to the methodsdescribed herein. By feeding ¹³C-enriched amino acids and simultaneouslyproviding intravenous supplementation of one or more amino acids, thelikely source of exogenous peptides not directly derived from wheatproteins can be identified. Those peptides of microbial origin in thegut will contain high ratios of ¹³C-amino acids. Lower stable isotopecontents, particularly of the intravenously administered amino acids,are indicative of peptides synthesized in the blood or in human tissues.The efficacy of peptidase or secretin supplementation can similarly beexplored with this technique.

Proving that exogenous peptides originating from the gut appear incerebral spinal fluid and identifying the ultimate source of thesepeptides can be used in appropriate clinical treatment of autism. Forexample, the stable isotope technique can be used in the development ofa rapid early diagnostic tool for clinicians, allowing for earlierclinical intervention before the effects of diet or yeast infectionbecome irreversible.

EXAMPLE 6

In this example a culture of Escherichia coli DH5α was grownexponentially at 37° C. on a [¹²C]-glucose morpholinopropanesulfonicacid buffered minimal media as described in Neidhardt et al., JBacteriol. 119:736 (1974), to a cell density of AU₆₀₀=0.7. At this celldensity 0.84 ml of a 500 mM solution of [¹³C]₆-glucose was added to 84ml of the culture, resulting in a approximate equimolar ratio of[¹³C]₆-glucose and [¹²C]₆-glucose. Approximately 8 ml aliquots werewithdrawn from the culture periodically and quenched in 2 ml ice coldtrichloroacetic acid (TCA) to a final concentration of 10% TCA byweight. The quenched samples were centrifuged at 4,000 rpm, 10° C., for40 min. Aliquots (1 ml) of the TCA supernatants were placed into 2 mlmicrofuge tubes and concentrated in a Savant Speed Vap to dryness. Thesamples were resuspended in HPLC-grade water to a total of 0.7 mlcombined from the resuspended concentrates for each timepoint sample.

The resuspended TCA soluble fractions were subjected to massspectrometric analysis in negative ion mode on a PE Biosystems Mariner™microelectrospray time-of-flight mass spectrometer. The massspectrometer was calibrated immediately prior to analysis per themanufacturer's instructions. Samples were fed continuously at 0.3 μl/mininto the microspray ionization system with a nozzle potential of 160 Vand nozzle temperature of 170° C. Mass spectra were accumulated forabout 15 min.

The mass spectrometric data were analyzed by dividing the countsdetermined exactly 6.02013 amu higher than each position by the peakcounts in each mass position, yielding a ¹³C/¹²C isotope ratio spectrumfor all six carbon species. The ¹³C/¹²C ratio spectra obtained for thesamples taken at each time point were plotted together and manuallyinspected for peaks that changed consistently over the timed samples. Inthis analysis it is expected that the zero time spectra would show peaksthat were significantly smaller than 1 and spectra from longer timepoints after substrate addition (i.e., after metabolism of the[¹³C]-glucose) would exhibit a ratio that asymptotically approached anequilibrium value closer to 1. Manual inspection of the ratio spectrashowed seven such potential six carbon metabolites (FIGS. 11 to 17).

Since the equilibrium substrate ratio was expected to be roughlyequimolar the actual MS spectra (scaled to the total counts) wereanalyzed at each of the corresponding ¹²C and ¹³C positions resultingfrom the ratio analysis. Three of the seven putative six carbonmetabolites were eliminated by this second level analysis because theputative ¹²C and ¹³C peaks were not found to be of the same magnitude(FIGS. 11, 15, and 17).

Finally, the metabolic flux was determined by curve fit to the equation:

${Flux}_{analyte} = \frac{\ln\left\{ \frac{\left( {{RA}_{t} - {RA}_{ss}} \right)}{\left( {{RA}_{o} - {RA}_{ss}} \right)} \right\}}{(t)\left( {{unit}\mspace{14mu}{of}\mspace{14mu}{sample}} \right)}$where RA_(SS) was neglected. Only the metabolites at 150.87 amu (FIG.12) and 152.88 amu (FIG. 13) were found to have metabolic flux valuessignificantly different from zero and exhibited roughly equimolar peaksat both the ¹³C and ¹²C positions at long times. These mass positionsare the most likely to correspond to real metabolites resulting directlyfrom the six glucose carbons, and thus correspond to a metabolic C₆fingerprint of glucose metabolism by E. coli at these growth conditions.The metabolic fluxes of the 150.87 and 152.88 amu metabolites were foundto be similar at about 1×10⁻² and 9×10⁻³ s⁻¹AU₆₀₀ ⁻¹, respectively.

A similar analysis was conducted for five carbon metabolites, whichsuggests that two five carbon metabolites may also be derived from[¹³C]-glucose metabolism (FIGS. 18 and 19). However, only in the longtime points does the 278.81 amu metabolite exhibit any significantincrease in ¹³C content (FIG. 18), suggesting that this peak may be anexperimental artifact. The 280.80 amu metabolite appears to exhibit areal flux of about 4×10⁻³ s⁻¹AU₆₀₀ ⁻¹, about half of that observed forthe six carbon metabolites. The identities of these metabolites isunknown.

Similar analyses were conducted at the four, three, and two carbonmetabolite levels with no apparent ¹³C-metabolites resulting from[¹³C]-glucose being found in the mass spectral data.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims. All publications, patents, and patentapplications cited herein are hereby incorporated by reference in theirentirety for all purposes to the same extent as if each individualpublication, patent or patent application were specifically andindividually indicated to be so incorporated by reference.

1. A method for analyzing a metabolic pathway, comprising: (a)administering to a subject a known ratio of a labeled substrate and anunlabeled substrate, wherein said labeled substrate is a substratelabeled with a stable isotope and said unlabeled substrate is saidsubstrate having a natural abundance of said stable isotope; (b)allowing the labeled substrate and the unlabeled substrate to be atleast partially metabolized by the subject to form a labeled targetmetabolite and an unlabeled target metabolite; (c) generating massspectral data from a plurality of samples from said subject at aplurality of time points; and (d) determining a plurality of relativeabundances of said labeled target metabolite to said unlabeled targetmetabolite from the mass spectral data of at least one of said samplesand comparing said plurality of relative abundances to said known ratioof said labeled substrate and said unlabeled substrate.
 2. The method ofclaim 1, further comprising at least partially separating the labeledtarget metabolites and the unlabeled target metabolites from otherbiological components in said at least one of said samples prior todetermining the plurality of relative abundances.
 3. The method of claim2, wherein the separating comprises performing a plurality of capillaryelectrophoresis methods in series.
 4. The method of claim 3, wherein theplurality of capillary electrophoresis methods are selected from thegroup consisting of capillary zone electrophoresis, capillaryisoelectric focusing and capillary gel electrophoresis.
 5. The method ofclaim 4, wherein the plurality of capillary electrophoresis methods areselected from the group consisting of capillary zone electrophoresis andcapillary isoelectric focusing.
 6. The method of claim 5, wherein theperforming of the capillary electrophoresis methods comprises performinga plurality of capillary zone electrophoresis methods.
 7. The method ofclaim 3, wherein the performing of the capillary electrophoresis methodsgenerate separate fractions for at least one class of metabolite,wherein the class of metabolite is selected from the group consisting ofproteins, polysaccharides, carbohydrates, nucleic acids, amino acids,nucleotides, nucleosides, fats, fatty acids and organic acids.
 8. Themethod of claim 3, wherein the separating comprises conducting anon-electrophoretic separation technique prior to conducting theplurality of electrophoresis methods to precipitate at least some of thebiological components.
 9. The method of claim 1, wherein the stableisotope is selected from the group consisting of ¹³C, ²H, ¹⁵N, ¹⁸O and³⁴S.
 10. The method of claim 1, wherein the substrate is selected fromthe group consisting of proteins, carbohydrates, nucleic acids, aminoacids, nucleotides, nucleosides, fatty acids, organic acids, and fats.11. The method of claim 10, wherein the substrate is a protein.
 12. Themethod of claim 1, wherein the plurality of samples are obtained from abodily fluid, the bodily fluid selected from the group consisting ofblood, urine, cerebral fluid, spinal fluid, sweat, and gastrointestinalfluids.
 13. The method of claim 1, wherein the plurality of samples arecells, tissue samples or fecal materials.